Alphavirus replicon vector systems

ABSTRACT

The present invention provides compositions useful in and methods for producing populations of infectious, replication-defective alphavirus replicon particles that contain no replication-competent alphavirus particles, as determined by passage on cells in culture. The compositions include helper and replicon nucleic acid molecules that can further reduce the predicted frequency for formation of replication-competent virus and can optimize manufacturing strategies and costs.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of priority from U.S. ProvisionalApplication No. 60/317,722, filed Sep. 6, 2001, which is hereinincorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to improved constructs for andmethods of making recombinant alphavirus particles that are useful inimmunotherapies for infectious diseases and cancer and in the deliveryof genes for therapeutic purposes.

BACKGROUND OF THE INVENTION

[0003] Alphaviruses are currently being used as a vector platform todevelop vaccines for infectious diseases (e.g. see U.S. Pat. Nos.5,792,462; 6,156,558; 5,811,407; 5,789,245; 6,015,694; 5,739,026; Pushkoet al., Virology 239(2): 389-401 (1997), Frolov et al., J. Virol. 71(1):248-258 (1997); Smerdou and Liljestrom, J. Virol. 73(2): 1092-1098(1999). Alphaviruses comprise a genus in the Togaviridae, and members ofthe genus are found throughout the world, in both vertebrate andinvertebrate hosts. Among the most studied alphaviruses for vectorplatforms are Venezuelan Equine Encephalitis (VEE) Virus, SemilikiForest Virus (SFV), and Sindbis Virus, the prototype member of thegenus. Various constructs have been developed to enhance immunogenicityand effectiveness in vaccine applications. Many of these constructs havealso been designed to decrease the likelihood of formation ofreplication-competent virus through recombination. Johnston et al. (U.S.Pat. Nos. 5,792,462 and 6,156,558, cited above) recognized the potentialfor recombination from a single helper system (in which the complete setof structural proteins of an alphavirus are on one RNA molecule and thenonstructural proteins and gene of interest are on another molecule),and thus designed “double-helper” systems that utilized two helper RNAsto encode the structural proteins. Dubensky et al. (U.S. Pat. No.5,789,245) and Polo et al. (U.S. Pat. No. 6,242,259) describe the use oftwo DNA alphavirus structural protein expression cassettes to packagealphavirus replicons or other alphavirus vectors. Liljestrom andcolleagues have presented data confirming that a “single helper system”will generate wild-type virus particles through recombination (Bergland,et al. 1993 Biotechnology 11(8): 916-920).

[0004] By distributing the viral genes among three nucleic acids, two ofwhich comprise the helper system, as in the above-described art, thetheoretical frequency of recombination that would create areplication-competent virus is reduced significantly relative to singlehelper systems. These existing systems include the use of the alphavirusRNA polymerase recognition signals, so that the helper systems can takeadvantage of the presence of the alphavirus replication machinery foramplification and efficient expression of helper functions. However, thepresence of the terminal recognition signals on the helper RNAs alsomeans that recombinants in which the helper constructs are incorporatedinto the termini of the replicon RNA by RNA recombination remainreplicable. It is also recognized (e.g. Liljestrom et al. U.S. Pat. No.6,190,666, Column 17, lines 45-48) that the capsid binding region ofnsP1 is required for the packaging of alphaviral RNA into virus orviral-like particles, and so removal of this region would result in thereduction of packaging (see also Levis et al. 1986 Cell 44:137 and Weisset al. 1989 J. Virol. 63:530).

[0005] Thus, in existing replicon systems, known packaging signals aretypically included in replicon RNAs and excluded from helper constructs.However, helper RNAs are nonetheless packaged or copackaged at a lowerfrequency (Lu and Silver (J. Virol Methods 2001, 91(1): 59-65), andhelper constructs with terminal recognition signals will be amplifiedand expressed in the presence of a replicon, and potentially yieldadditional recombination events.

[0006] The current preferred dosages for administration of vectorreplicon particles, as described by Johnston et al., or recombinantalphavirus particles, as described by Dubensky et al., are approximately10⁶ to 10⁸ particles. In the case of chimpanzee administrations,Dubensky et al. have estimated the need for 4 injections, eachcontaining 10⁷-10⁸ particles, with a Sindbis-HBV vaccine. Such dosagesrequire large scale manufacturing procedures, and the amounts producedat such scale may be greater than the predicted frequency for thegeneration of replication-competent viruses in these existing systems.

[0007] Thus, there remains a need to further improve systems formanufacturing alphavirus replicon particles to further reduce thepredicted frequency for formation of replication-competent virus, and tooptimize manufacturing strategies and costs.

SUMMARY OF THE INVENTION

[0008] The present invention provides improved alphavirus repliconvector systems for producing infectious, replication defective,alphavirus replicon particles in alphavirus-permissive cells.Encompassed in the invention are improved replicon RNAs and improvedhelper nucleic acids for expressing the alphavirus structural proteins.During the production of recombinant alphavirus particles, thegeneration of replication-competent virus particles can occur throughrecombination alone or through a combination of helper packaging andrecombination. Thus, constructs are provided that eliminate or minimizethe occurrence of one or both of these events. In addition, theseconstructs are also designed to minimize the manufacturing complexityand cost of particles made with such constructs. The invention alsoprovides methods of making recombinant alphavirus particles using theclaimed constructs, and pharmaceutical compositions comprising theserecombinant alphavirus particles.

[0009] In a first aspect, resolving DNA helpers are provided, namelyrecombinant DNA molecules for expressing the alphavirus structuralproteins which comprise a promoter directing transcription of RNA from aDNA sequence comprising, in order (i) a first nucleic acid sequenceencoding at least one alphavirus structural protein, (ii) a secondnucleic acid sequence encoding a ribozyme, (iii) a third nucleic acidsequence encoding an IRES, and (iv) a fourth nucleic acid sequenceencoding at least one alphavirus structural protein, wherein at leastone alphavirus structural protein encoded by the fourth nucleic acidsequence is not encoded by the first nucleic acid sequence.

[0010] In another embodiment of the resolving DNA helpers of thisinvention, recombinant DNA molecules for expressing alphavirusstructural proteins are provided, comprising a promoter directing thetranscription of RNA from a DNA sequence comprising, in order: (i) afirst nucleic acid sequence encoding a 5′ alphavirus replicationrecognition sequence, (ii) a second nucleic acid sequence encodingeither (a) an RNA sequence that promotes transcription of a proteincoding RNA sequence or (b) an IRES; (iii) a third nucleic acid sequenceencoding at least one alphavirus structural protein, (iv) a fourthnucleic acid sequence encoding a 3′ alphavirus replication recognitionsequence, (v) a fifth nucleic acid sequence encoding a ribozyme, (vi) asixth nucleic acid sequence encoding an IRES, and (vii) a seventhnucleic acid sequence encoding at least one alphavirus structuralprotein, wherein at least one alphavirus structural protein encoded bythe seventh nucleic acid sequence is not encoded by the third nucleicacid sequence.

[0011] In yet another embodiment of the resolving DNA helpers, thepresent invention provides a recombinant DNA molecule for expressingalphavirus structural proteins comprising a promoter directing thetranscription of RNA from a DNA sequence comprising, in order: (i) afirst nucleic acid sequence encoding an IRES, (ii) a second nucleic acidsequence encoding at least one alphavirus structural protein, (iii)athird nucleic acid sequence encoding a ribozyme, (iv) a fourth nucleicacid sequence encoding an IRES, and (v) a fifth nucleic acid sequenceencoding at least one alphavirus structural protein, wherein at leastone alphavirus structural protein encoded by the fifth nucleic acidsequence is not encoded by the second nucleic acid sequence.

[0012] In a second aspect of the invention, methods for producinginfectious, replication-defective alphavirus replicon particlescomprising introducing into a population of alphavirus-permissive cellsone or more resolving DNA helper(s) of the claimed invention and analphavirus replicon RNA encoding at least one heterologous RNA such thatinfectious, replication-defective particles are produced.

[0013] In a third aspect, the present invention provides resolving RNAhelpers, namely recombinant DNA molecules for expressing alphavirusstructural proteins comprising: (i) a DNA dependent RNA polymerasepromoter, (ii) an IRES, (iii) a nucleic acid sequence encoding analphavirus capsid protein, which is modified to remove the active siteof the autoprotease, (iv) a non-autocatalytic protease recognition site,and (v) a nucleic acid sequence encoding at least one alphavirusglycoprotein.

[0014] In another embodiment of the resolving RNA helpers of thisinvention, a recombinant DNA molecule for expressing a resolving RNAhelper in vivo is provided comprising (i) a DNA dependent RNA polymerasepromoter, (ii) a nucleic acid sequence encoding at least one alphavirusstructural protein, (iii) a non-autocatalytic protease recognition site,and (iv) a nucleic acid sequence encoding at least one alphavirusstructural protein, wherein the nucleic acid sequences in (ii) and (iv)are not identical. In preferred embodiments of this aspect, the promoteris an RNA polymerase II promoter that is operable in a helper cell.

[0015] In yet another embodiment of the resolving RNA helpers of thisinvention, a recombinant DNA molecule for expressing an RNA helper invitro is provided, comprising a promoter directing the transcription ofRNA from a DNA sequence comprising (i) a first nucleic acid sequenceencoding an alphavirus 5′ replication recognition sequence, (ii) atranscriptional promoter, (iii) a nucleic acid sequence encoding atleast one alphavirus structural protein, (iv) a non-autocatalyticprotease recognition site, and (v) a nucleic acid sequence encoding atleast one alphavirus structural protein, and (vi) an alphavirus 3′replication recognition sequence, wherein the nucleic acid sequences of(iii) and (v) are not identical.

[0016] In a fourth aspect, methods for producing infectious,replication-defective alphavirus replicon particles comprisingintroducing into a population of cells (i) one or more resolving RNAhelpers of the present invention, (ii) a protease that recognizes thenon-autocatalytic protease recognition site, and (iii) an alphavirusreplicon RNA encoding at least one heterologous RNA such thatinfectious, replication-defective alphavirus replicon particles areproduced in the cells. In certain embodiments of this aspect, an RNApolymerase that recognizes the DNA dependent RNA polymerase promoter isalso made available in the helper cell along with the recombinant DNAmolecule, such that the DNA molecule is transcribed in vivo to producesufficient alphavirus structural proteins for packaging alphavirusreplicon particles.

[0017] In a fifth aspect of the present invention, a rearrangedalphavirus RNA replicon vector is provided comprising in order: (i) afirst nucleic acid sequence encoding a 5′ alphavirus replicationrecognition sequence, (ii) a second nucleic acid encoding alphavirusnonstructural proteins nsp1, nsp2, and nsp3; (iii) either (a) atranscriptional promoter or (b) an IRES, (iv) a nucleic acid encoding atleast one heterologous gene of interest, (v) an IRES, (vi) a thirdnucleic acid encoding an alphavirus nonstructural protein nsp4, and(vii) a fourth nucleic acid encoding a 3′ alphavirus replicationrecognition sequence. In another embodiment of this aspect, a vectorconstruct comprising a 5′ promoter operably linked to a cDNA of therearranged alphavirus replicon RNA is provided.

[0018] In a sixth aspect, compositions comprising a population ofinfectious, defective, alphavirus particles, wherein each particlecontains an alphavirus replicon RNA comprising a rearranged alphavirusreplicon RNA of this invention, and the population has no detectablereplication-competent virus, as measured by passage on cell cultures.

[0019] In a seventh aspect, a non-replicating DNA helper is provided,namely a recombinant DNA molecule for expressing alphavirus structuralproteins comprising a promoter for directing the transcription of RNAfrom a DNA sequence operably linked to a DNA sequence encoding acomplete alphavirus structural polyprotein-coding sequence, with theproviso that the DNA sequence does not encode alphaviral 5′ or 3′replication recognition sequences or an alphavirus subgenomic promoter.

[0020] In an eighth aspect, a chimeric alphavirus RNA helper isprovided, namely a recombinant RNA molecule comprising, in order, a 5′alphavirus replication recognition sequence, a promoter, a nucleic acidencoding at least one alphavirus structural protein, and a 3′ alphaviralreplication recognition sequence, wherein the promoter is operablylinked to the nucleic acid sequence encoding at least one alphavirusstructural protein, wherein the transcription-initiating sequence andthe RNA polymerase recognition sequence are recognized by thenonstructural viral proteins of Venezuelan equine encephalitis virus,and wherein the transcription-initiating sequence and the RNA polymeraserecognition sequence are derived from a virus other than the alphavirusencoding the structural protein.

[0021] In a ninth aspect, an alphavirus structural protein expressionsystem based on a virus other than an alphavirus is provided, comprisingtwo RNA molecules, wherein (a) a first RNA encodes sequence for viralreplicase proteins, and (b) a second recombinant RNA encodes sequencesfor (i) the 5′ replication recognition sequence for a replicationcomplex comprising the viral replicase proteins of (a), (ii) one or morealphavirus structural proteins, and (iii) the 3′ replication recognitionsequence for the replication complex comprising the viral replicaseproteins of (a), wherein, when the first RNA and the second RNA areintroduced into a helper cell, the first RNA replicates the second RNA,then the second RNA is translated to produce one or more alphavirusstructural proteins. In preferred embodiments of this aspect, therecombinant RNAs and the viral replicase proteins are derived from anodavirus.

[0022] In a tenth aspect of the invention, methods for producinginfectious, replication-defective alphavirus replicon particlescomprising introducing into a population of alphavirus-permissive cellsone or more helper nucleic acids selected from the group consisting ofnon-replicating DNA helpers, chimeric alphavirus helpers, and anon-alphavirus based helper system and an alphavirus replicon RNAencoding at least one heterologous RNA, such that the helpers expressall of the alphavirus structural proteins, producing said alphavirusreplicon particles in the cells, and collecting said alphavirus repliconparticles from the cell.

[0023] In an eleventh aspect of this invention, helper cells forexpressing infectious, replication-defective, alphavirus particlesutilizing any combination of the helpers disclosed hereinabove areprovided comprising, in an alphavirus-permissive cell, (i) one or morerecombinant nucleic acid molecules selected from group consisting of theresolving DNA helpers, the resolving RNA helpers, the non-replicatingDNA helpers, the chimeric alphavirus helpers and the non-alphavirushelper system, and (ii) an alphavirus replicon RNA encoding at least oneheterologous RNA, wherein the one or more recombinant nucleic acidhelpers together encode all alphavirus structural proteins whichassemble together into the alphavirus replicon particles.

DETAILED DESCRIPTION OF THE INVENTION

[0024] Definitions

[0025] As used herein, the term “alphavirus” has its conventionalmeaning in the art, and includes the various species such as VEE, SFV,Sindbis, Ross River Virus, Western Equine Encephalitis Virus, EasternEquine Encephalitis Virus, Chikungunya, S.A. AR86, Everglades virus,Mucambo, Barmah Forest Virus, Middelburg Virus, Pixuna Virus,O'nyong-nyong Virus, Getah Virus, Sagiyama Virus, Bebaru Virus, MayaroVirus, Una Virus, Aura Virus, Whataroa Virus, Banbanki Virus, KyzylagachVirus, Highlands J Virus, Fort Morgan Virus, Ndumu Virus, and BuggyCreek Virus. The preferred alphaviruses used in the constructs andmethods of the claimed invention are VEE, S.AAR86, Sindbis (e.g. TR339,see U.S. Pat. No. 6,008,035), and SFV.

[0026] The terms “5′ alphavirus replication recognition sequence” and“3′ alphavirus replication recognition sequence” refer to the sequencesfound in alphaviruses, or sequences derived therefrom, that arerecognized by the nonstructural alphavirus replicase proteins and leadto replication of viral RNA. These are sometimes referred to as the 5′and 3′ ends, or alphavirus 5′ and 3′ sequences. In the constructs ofthis invention, the use of these 5′ and 3′ ends will result inreplication of the RNA sequence encoded between the two ends. Thesesequences can be modified by standard molecular biological techniques tofurther minimize the potential for recombination or to introduce cloningsites, with the proviso that they must still be recognized by thealphavirus replication machinery.

[0027] The term “minimal 5′ alphavirus replication recognition sequence”refers to the minimal sequence that allows recognition by thenonstructural proteins of the alphavirus but does not result insignificant packaging/recombination of RNA molecules containing thesequence. In a preferred embodiment, the minimal 5′ alphavirusreplication recognition sequence results in a fifty to one-hundred folddecrease in the observed frequency of packaging/recombination of the RNAcontaining that sequence. Packaging/recombination of helpers can beassessed by several methods, e.g. the method described by Lu and Silver(J. Virol Methods 2001, 91(1): 59-65).

[0028] The terms “alphavirus RNA replicon”, “alphavirus replicon RNA”and “alphavirus RNA vector replicon” are used interchangeably to referto an RNA molecule expressing nonstructural protein genes such that itcan direct its own replication (amplification) and comprises, at aminimum, the 5′ and 3′ alphavirus replication recognition sequences,coding sequences for alphavirus nonstructural proteins, and apolyadenylation tract. It may additionally contain a promoter or anIRES. It may also be engineered to express alphavirus structuralproteins. Johnston et al. and Polo et al. (cited in the background)describe numerous constructs for such alphavirus RNA replicons, and suchconstructs are incorporated herein by reference. Specific embodiments ofthe alphavirus RNA replicons utilized in the claimed invention maycontain one or more attenuating mutations, an attenuating mutation beinga nucleotide deletion, addition, or substitution of one or morenucleotide(s), or a mutation that comprises rearrangement or chimericconstruction which results in a loss of virulence in a live viruscontaining the mutation as compared to the appropriate wild-typealphavirus. Examples of an attenuating nucleotide substitution(resulting in an amino acid change in the replicon) include a mutationat nsP1 amino acid position 538, nsP2 amino acid position 96, or nsP2amino acid position 372 in the alphavirus S.A.AR86.

[0029] The terms “alphavirus structural protein/protein(s)” refers toone or a combination of the structural proteins encoded by alphaviruses.These are produced by the virus as a polyprotein and are representedgenerally in the literature as C-E3-E2-6k-E1. E3 and 6k serve asmembrane translocation/transport signals for the two glycoproteins, E2and E1. Thus, use of the term E1 herein can refer to E1, E3-E1, 6k-E1,or E3-6k-E1, and use of the term E2 herein can refer to E2, E3-E2,6k-E2, or E3-6k-E2.

[0030] The term “helper(s)” refers to a nucleic acid molecule that iscapable of expressing one or more alphavirus structural proteins.

[0031] The terms “helper cell” and “packaging cell” are usedinterchangeably herein and refer to the cell in which alphavirusreplicon particles are produced. The helper cell comprises a set ofhelpers that encode one or more alphavirus structural proteins. Asdisclosed herein, the helpers may be RNA or DNA. The cell can be anycell that is alphavirus-permissive, i.e. cells that are capable ofproducing alphavirus particles upon introduction of a viral RNAtranscript. Alphavirus-permissive cells include, but are not limited to,Vero, baby hamster kidney (BHK), 293, 293T, chicken embryo fibroblast(CEF), and Chinese hamster ovary (CHO) cells. In certain embodiments ofthe claimed invention, the helper or packaging cell may additionallyinclude a heterologous RNA-dependent RNA polymerase and/or asequence-specific protease.

[0032] The terms “alphavirus replicon particles”, “virus repliconparticles” or “recombinant alphavirus particles”, used interchangeablyherein, mean a virion-like structural complex incorporating analphavirus replicon RNA that expresses one or more heterologous RNAsequences. Typically, the virion-like structural complex includes one ormore alphavirus structural proteins embedded in a lipid envelopeenclosing a nucleocapsid that in turn encloses the RNA. The lipidenvelope is typically derived from the plasma membrane of the cell inwhich the particles are produced. Preferably, the alphavirus repliconRNA is surrounded by a nucleocapsid structure comprised of thealphavirus capsid protein, and the alphavirus glycoproteins are embeddedin the cell-derived lipid envelope. The alphavirus replicon particlesare infectious but replication-defective, i.e. the replicon RNA cannotreplicate in the host cell in the absence of the helper nucleic acid(s)encoding the alphavirus structural proteins.

[0033] As described in detail hereinbelow, the present inventionprovides improved alphavirus-based replicon systems that reduce thepotential for replication-competent virus formation and that aresuitable and/or advantageous for commercial-scale manufacture ofvaccines or therapeutics comprising them. The present invention providesimproved alphavirus RNA replicons and improved helpers for expressingalphavirus structural proteins.

[0034] In one embodiment of this invention, a series of “helperconstructs”, i.e. recombinant DNA molecules that express the alphavirusstructural proteins, is disclosed in which a single helper isconstructed that will resolve itself into two separate molecules invivo. Thus, the advantage of using a single helper in terms of ease ofmanufacturing and efficiency of production is preserved, while theadvantages of a bipartite helper system are captured in the absence ofemploying a bipartite expression system. In one set of theseembodiments, a DNA helper construct is used, while in a second set anRNA helper vector is used. In the case of the DNA helper constructs thatdo not employ alphaviral recognition signals for replication andtranscription, the theoretical frequency of recombination is lower thanthe bipartite RNA helper systems that employ such signals.

[0035] In the preferred embodiments for the constructs of thisinvention, a promoter for directing transcription of RNA from DNA, i.e.a DNA dependent RNA polymerase, is employed. In the RNA helperembodiments, the promoter is utilized to synthesize RNA in an in vitrotranscription reaction, and specific promoters suitable for this useinclude the SP6, T7, and T3 RNA polymerase promoters. In the DNA helperembodiments, the promoter functions within a cell to directtranscription of RNA. Potential promoters for in vivo transcription ofthe construct include eukaryotic promoters such as RNA polymerase IIpromoters, RNA polymerase III promoters, or viral promoters such as MMTVand MoSV LTR, SV40 early region, RSV or CMV. Many other suitablemammalian and viral promoters for the present invention are available inthe art. Alternatively, DNA dependent RNA polymerase promoters frombacteria or bacteriophage, e.g. SP6, T7, and T3, may be employed for usein vivo, with the matching RNA polymerase being provided to the cell,either via a separate plasmid, RNA vector, or viral vector. In aspecific embodiment, the matching RNA polymerase can be stablytransformed into a helper cell line under the control of an induciblepromoter. Constructs that function within a cell can function asautonomous plasmids transfected into the cell or they can be stablytransformed into the genome. In a stably transformed cell line, thepromoter may be an inducible promoter, so that the cell will onlyproduce the RNA polymerase encoded by the stably transformed constructwhen the cell is exposed to the appropriate stimulus (inducer). Thehelper constructs are introduced into the stably transformed cellconcomitantly with, prior to, or after exposure to the inducer, therebyeffecting expression of the alphavirus structural proteins.Alternatively, constructs designed to function within a cell can beintroduced into the cell via a viral vector, e.g. adenovirus, poxvirus,adeno-associated virus, SV40, retrovirus, nodavirus, picornavirus,vesicular stomatitis virus, and baculoviruses with mammalian pol IIpromoters.

[0036] Once an RNA transcript (mRNA) encoding the helper or RNA repliconvectors of this invention is present in the helper cell (either via invitro or in vivo approaches, as described above), it is translated toproduce the encoded polypeptides or proteins. The initiation oftranslation from an MRNA involves a series of tightly regulated eventsthat allow the recruitment of ribosomal subunits to the mRNA. Twodistinct mechanisms have evolved in eukaryotic cells to initiatetranslation. In one of them, the methyl-7-G(5′)pppN structure present atthe 5′ end of the mRNA, known as “cap”, is recognized by the initiationfactor eIF4F, which is composed of eIF4E, eIF4G and eIF4A. Additionally,pre-initiation complex formation requires, among others, the concertedaction of initiation factor eIF2, responsible for binding to theinitiator tRNA-Met₁, and eIF3, which interacts with the 40S ribosomalsubunit (reviewed in Hershey & Merrick. Translational Control of GeneExpression, pp. 33-88. Cold Spring Harbor, N.Y.: Cold Spring HarborLaboratory Press. 2000.)

[0037] In the alternative mechanism, translation initiation occursinternally on the transcript and is mediated by a cis-acting element,known as an internal ribosome entry site (IRES), that recruits thetranslational machinery to an internal initiation codon in the mRNA withthe help of trans-acting factors (reviewed in Jackson. TranslationalControl of Gene Expression, pp. 127-184. Cold Spring Harbor LaboratoryPress. 2000). During many viral infections, as well as in other cellularstress conditions, changes in the phosphorylation state of eIF2, whichlower the levels of the ternary complex eIF2-GTP-tRNA-Met₁, results inoverall inhibition of protein synthesis. Conversely, specific shut-offof cap-dependent initiation depends upon modification of eIF4Ffunctionality (Thompson & Sarnow, Current Opinion in Microbiology 3,366-370, 2000).

[0038] IRES elements bypass cap-dependent translation inhibition; thusthe translation directed by an IRES is termed “cap-independent”. Hence,IRES-driven translation initiation prevails during many viralinfections, for example picornaviral infection (Macejak & Sarnow. Nature353, 90-94, 1991). Under these circumstances, cap-dependent initiationis inhibited or severely compromised due to the presence of smallamounts of functional eIF4F. This is caused by cleavage or loss ofsolubility of eIF4G (Gradi et al., Proceedings of the National Academyof Sciences, USA 95, 11089-11094, 1998); 4E-BP dephosphorylation(Gingras et al., Proceedings of the National Academy of Sciences, USA93, 5578-5583. 1996) or poly(A)-binding protein (PABP) cleavage(Joachims et al., Journal of Virology 73, 718-727, 1999).

[0039] IRES sequences have been found in numerous transcripts fromviruses that infect vertebrate and invertebrate cells as well as intranscripts from vertebrate and invertebrate genes. Examples of IRESelements suitable for use in this invention include: viral IRES elementsfrom Picornaviruses e.g. poliovirus (PV), encephalomyocarditis virus(EMCV), foot-and-mouth disease virus (FMDV), from Flaviviruses e.g.hepatitis C virus (HCV), from Pestiviruses e.g. classical swine fevervirus (CSFV), from Retroviruses e.g. murine leukemia virus (MLV), fromLentiviruses e.g. simian immunodeficiency virus (SIV), or cellular mRNAIRES elements such as those from translation initiation factors e.g.eIF4G or DAP5, from Transcription factors e.g. c-Myc (Yang and Sarnow,Nucleic Acids Research 25: 2800-2807 1997) or NF-κ-repressing factor(NRF), from growth factors e.g. vascular endothelial growth factor(VEGF), fibroblast growth factor (FGF-2), platelet-derived growth factorB (PDGF B), from homeotic genes e.g. Antennapedia, from survivalproteins e.g. X-Linked inhibitor of apoptosis (XIAP) or Apaf-1, orchaperones e.g. the immunoglobulin heavy-chain binding protein BiP(reviewed in Martinez-Salas et al., Journal of General Virology. 82:973-984, 2001.)

[0040] Preferred IRES sequences that can be utilized in theseembodiments are derived from: encephalomyocarditis virus (EMCV,accession # NC001479), cricket paralysis virus (accession # AF218039),Drosophila C virus accession # AF014388, Plautia stali intestine virus(accession # AB006531), Rhopalosiphum padi virus (accession # AF022937),Himetobi P virus (accession # AB017037), acute bee paralysis virus(accession # AF150629), Black queen cell virus (accession # AF183905),Triatoma virus (accession # AF178440), Acyrthosiphon pisu virus(accession # AF024514), infectious flacherie virus (accession #AB000906), and Sacbrood virus (accession # AF092924). In addition to thenaturally occurring IRES elements listed above, synthetic IRESsequences, designed to mimic the function of naturally occurring IRESsequences, can also be used. In the embodiments in which an IRES is usedfor translation of the promoter driven constructs, the IRES may be aninsect TRES or another non-mammalian IRES that is expressed in the cellline chosen for packaging of the recombinant alphavirus particles, butwould not be expressed, or would be only weakly expressed, in the targethost. In those embodiments comprising two IRES elements, the twoelements may be the same or different.

[0041] Rearranged Alphavirus RNA Replicon Vectors

[0042] In all systems described to date which employ alphavirus RNAvector replicons to express a heterologous gene of interest, the portionof the alphavirus genome that encodes the alphavirus nonstructuralproteins (nsps) is maintained intact, i.e. it appears in the repliconvectors exactly as it appears in the alphavirus. Disclosed herein is arearranged alphavirus RNA replicon vector in which the sequence encodingnsp4 has been separated from the sequence encoding nsps1-3 and placedunder the control of a separate translational control element, such asan IRES. Although it is under separate control and displaced from theother nonstructural coding sequences, the sequence encoding nsp4 istranscribed from the incoming, plus strand of the virus, so that allnonstructural proteins are produced when the replicon is introduced intothe helper cell. The cassette directing expression of the heterologousgene of interest is placed between the two nonstructural gene sequences,which together encode all of the alphavirus nonstructural proteins.

[0043] In the existing tripartite alphavirus replicon systems (twohelper molecules and the vector replicon), recombination between thehelper(s) and the replicon is required to generate areplication-competent virus. In these systems, it is generally thoughtthat the viral polymerase complex moves between the helper molecules andthe replicon molecule (“strand-switching”), replicating sequences fromall three molecules. In these systems, the strand-switching may occuranywhere in the replicon 3′ to the 26S promoter directing expression ofthe heterogogous RNA, and still replicate all of the nonstructuralpolyprotein coding region “nsps1-4”. In the rearranged replicons claimedherein, the theoretical frequency of generation of areplication-competent virus is much lower, since any recombination intothe heterologous gene region results in the loss of the downstream nsp4gene in the recombinant.

[0044] Thus, described herein is a recombinant nucleic acid comprising,in order: (i) a first nucleic acid sequence encoding a 5′ alphavirusreplication recognition sequence, (ii) a second nucleic acid encodingalphavirus nonstructural proteins nsp 1, nsp 2, and nsp3; (iii) an IRES,(iv) a nucleic acid sequence encoding at least one heterologous gene ofinterest, (v) an IRES, (vi) a third nucleic acid encoding the alphavirusnonstructural protein nsp4, and (vii) a fourth nucleic acid encoding a3′ alphavirus replication recognition sequence. In another embodiment,element (iii) is a transcriptional promoter, such as the alphavirussubgenomic promoter (also referred to as the 26S promoter or the viraljunction region promoter). In certain embodiments, this vector repliconRNA is transcribed in vitro from a DNA plasmid and then introduced intothe helper cell by electroporation. In other embodiments, the vectorreplicon RNA of this invention is transcribed in vivo from a DNA vectorplasmid that is transfected into the helper cell (e.g. see U.S. Pat. No.5,814,482), or it is delivered to the helper cell via a virus orvirus-like particle.

[0045] The heterologous gene of interest, also referred to herein as aheterologous RNA or heterologous sequence, can be chosen from a widevariety of sequences derived from viruses, prokaryotes or eukaryotes.Examples of categories of heterologous sequences include, but are notlimited to, immunogens, cytokines, toxins, therapeutic proteins,enzymes, antisense sequences, and immune response modulators.

[0046] In a preferred embodiment, the 3′ alphavirus non-coding sequenceused in the replicon construct is approximately 300 nucleotides inlength, which contains the 3′ replication recognition sequence. Theminimal 3′ replication recognition sequence, conserved amongalphaviruses, is a 19 nucleotide sequence (Hill et al., Journal ofVirology, 2693-2704, 1997). The 3′ non-coding sequence can be modifiedthrough standard molecular biological techniques to minimize the size ofthe 3′ end while preserving the replication function.

[0047] Resolving DNA Helpers

[0048] Several specific embodiments of the resolving DNA helperconstructs are disclosed hereinbelow. In one embodiment, this inventiondiscloses a recombinant DNA molecule for expressing alphavirusstructural proteins comprising a promoter directing the transcription ofRNA from a DNA sequence comprising, in order: (i) a first nucleic acidsequence encoding at least one alphavirus structural protein, (ii) asecond nucleic acid sequence encoding a ribozyme, (iii) a third nucleicacid sequence encoding an IRES, and (iv) a fourth nucleic acid sequenceencoding at least one alphavirus structural protein, wherein at leastone alphavirus structural protein encoded by the fourth nucleic acidsequence is not encoded by the first nucleic acid sequence. In apreferred embodiment, the promoter is a pol II promoter, such as the CMVpromoter.

[0049] In a further specific embodiment thereof, the first nucleic acidsequence is selected from the group of nucleic acid sequences encoding:capsid, E1 glycoprotein, E2 glycoprotein, E1 and E2 glycoprotein, capsidand E1 glycoprotein, or capsid and E2 glycoprotein. In these specificembodiments, the structural gene nucleic acid sequence(s) encoded by thefourth nucleic acid sequence is selected from this same group. In apreferred embodiment, the combination of sequences encoded by the firstand fourth nucleic acid sequences encompass all the structural proteinsrequired to assemble a recombinant alphavirus particle. In a furtherspecific embodiment, one or more of the alphavirus structural proteinsmay encode one or more attenuating mutations, for example as defined inU.S. Pat. Nos. 5,792,462 and 6,156,558. Specific attenuating mutationsfor the VEE E1 glycoprotein include an attenuating mutation at any oneof E1 amino acid positions 81, 272 or 253. Alphavirus replicon particlesmade from the VEE-3042 mutant contain an isoleucine substitution atE1-81, and virus replicon particles made from the VEE-3040 mutantcontain an attenuating mutation at E1-253. Specific attenuatingmutations for the VEE E2 glycoprotein include an attenuating mutation atany one of E2 amino acid positions 76, 120, or 209. Alphavirus repliconparticles made from the VEE-3014 mutant contain attenuating mutations atboth E1-272 and at E2-209 (see U.S. Pat. No. 5,792,492). A specificattenuating mutation for the VEE E3 glycoprotein includes an attenuatingmutation consisting of a deletion of E3 amino acids 56-59. Virusreplicon particles made from the VEE-3526 mutant contain this deletionin E3 (aa56-59) as well as a second attenuating mutation at E1-253.Specific attenuating mutations for the S.A.AR86 E2 glycoprotein includean attenuating mutation at any one of E2 amino acid positions 304, 314,372, or 376.

[0050] In another embodiment, this invention discloses a recombinant DNAmolecule for expressing alphavirus structural proteins comprising apromoter directing the transcription of RNA from a DNA sequencecomprising, in order: (i) a first nucleic acid sequence encoding a 5′alphavirus replication recognition sequence, (ii) a second nucleic acidsequence encoding either (a) an RNA sequence that promotes transcriptionof a protein coding RNA sequence or (b) an IRES; (iii) a third nucleicacid sequence encoding at least one alphavirus structural protein, (iv)a fourth nucleic acid sequence encoding a 3′ alphavirus replicationrecognition sequence, (v) a fifth nucleic acid sequence encoding aribozyme, (vi) a sixth nucleic acid sequence encoding an IRES, and (vii)a seventh nucleic acid sequence encoding at least one alphavirusstructural protein, wherein at least one alphavirus structural proteinencoded by the seventh nucleic acid sequence is not encoded by the thirdnucleic acid sequence. In a preferred embodiment, the promoter is a polII promoter, such as the CMV promoter. In another embodiment, thepromoter is the T7 promoter, and the T7 polymerase is provided in thepackaging cell in any one of the methods described hereinabove. In aspecific embodiment thereof, the third nucleic acid sequence is selectedfrom the group of nucleic acid sequences encoding: capsid, E1glycoprotein, E2 glycoprotein, E1 and E2 glycoprotein, capsid and E1glycoprotein, or capsid and E2 glycoprotein. In these specificembodiments, the structural gene nucleic acid sequence(s) encoded by theseventh nucleic acid sequence is selected from this same group. In apreferred embodiment thereof, the combination of sequences encoded bythe third and seventh nucleic acid sequences encompass all thestructural proteins required to assemble a recombinant alphavirusparticle. In a further specific embodiment, the sequences encoded by thethird nucleic acid comprise one or more of the alphavirus glycoproteingenes, and the sequence encoded by the seventh nucleic acid comprisesthe alphavirus capsid gene. In a further specific embodiment, one ormore of the alphavirus structural proteins may encode one or moreattenuating mutations, as defined in U.S. Pat. Nos. 5,792,462 and6,156,558, and specific examples of which are listed hereinabove.

[0051] In another embodiment, this invention discloses a recombinant DNAmolecule for expressing alphavirus structural proteins comprising apromoter directing the transcription of RNA from a DNA sequencecomprising, in order: (i) a first nucleic acid sequence encoding anIRES, (ii) a second nucleic acid sequence encoding at least onealphavirus structural protein, (iii) a third nucleic acid sequenceencoding a ribozyme, (iv) a fourth nucleic acid sequence encoding anIRES, and (v) a fifth nucleic acid sequence encoding at least onealphavirus structural protein, wherein at least one alphavirusstructural protein encoded by the fifth nucleic acid sequence is notencoded by the second nucleic acid sequence. In a preferred embodiment,the promoter is a pol II promoter, such as the CMV promoter. In anotherembodiment, the promoter is the T7 promoter, and the T7 polymerase isprovided in the packaging cell in any one of the methods describedhereinabove. In a specific embodiment thereof, the second nucleic acidsequence is selected from the group of nucleic acid sequences encoding:capsid, E1 glycoprotein, E2 glycoprotein, E1 and E2 glycoprotein, capsidand E1 glycoprotein, or capsid and E2 glycoprotein. In these specificembodiments, the structural gene nucleic acid sequence(s) encoded by thefifth nucleic acid sequence is selected from this same group. In apreferred embodiment thereof, the combination of sequences encoded bythe second and fifth nucleic acid sequences encompass all the structuralproteins required to assemble a recombinant alphavirus particle. In afurther specific embodiment, one or more of the alphavirus structuralproteins may encode one or more attenuating mutations, as defined inU.S. Pat. Nos. 5,792,462 and 6,156,558, and specific examples of whichare listed hereinabove.

[0052] As described above, the resolving ability of the DNA helperconstructs derives from the insertion of a ribozyme. Ribozymes arecatalytic RNA molecules possessing the ability to specifically catalyzeits own (cis-) or other (trans-) single-stranded RNA excision (cleavage)following transcription of the RNA in vivo from the DNA vector.Ribozymes target a specific RNA sequence, and different ribozymes targetdifferent RNA sequences. Through the insertion of nucleotide sequencesencoding these ribozymes into the DNA vector, it is possible to engineermolecules that will recognize specific nucleotide sequences within anRNA transcript and cleave it (Cech, T. Amer. Med. Assn., 260:3030,1988). When a single DNA helper construct as described herein isintroduced into a packaging cell, the ribozyme cleaves the single RNAtranscript synthesized in vivo from the introduced DNA construct at theribozyme target sequence, resulting in the generation of two separateRNA molecules within the cell, each encoding one or more than onealphaviral structural proteins.

[0053] A wide variety of ribozymes may be utilized within the context ofthe present invention, including for example, Group I intron ribozymes(Cech et al., U.S. Pat. No. 4,987,071); Group II Introns (Michel, etal., EMBO J. 2:33-38 1983), hairpin ribozymes (Hampel et al., Nucl.Acids Res. 18:299-304, 1990, U.S. Pat. No. 5,254,678 and European PatentPublication No. 0 360 257), hammerhead ribozymes (Rossi, J. J. et al.,Pharmac. Ther. 50:245-254, 1991; Forster and Symons, Cell 48:211-220,1987; Haseloff and Gerlach, Nature 328:596-600, 1988; Walbot andBruening, Nature 334:196, 1988; Haseloff and Gerlach, Nature 334:585,1988), hepatitis delta virus ribozymes (Perrotta and Been, Biochem.31:16, 1992); Neurospora Vakrud satellite (VS) ribozymes (Anderson andCollins, Mol. Cell 5: 4690478, 2000, RNase P ribozymes (Takada et al.,Cell 35:849, 1983); as well as other types of ribozymes (see e.g., WO95/29241, and WO 95/31551). Further examples of ribozymes include thosedescribed in U.S. Pat. Nos. 5,116,742, 5,225,337 and 5,246,921.

[0054] The Group I intron ribozyme was the first known ribozyme whichwas described by Cech and colleagues in Tetrahymena in 1982 (Kruger etal. Cell 31: 147-157, 1992). This ribozyme was found to be involved inthe processing of ribosomal RNA (rRNA) through a unique self-splicingmanner. The self-splicing of rRNA occurs by a two step mechanism. First,a guanine nucleotide is added to the 5′ end of the intron as theintron-exon junction is being cleaved. Then the freed 5′ intron withguanine attacks at the 3′ intron-exon junction to release the intron andgenerate spliced exons (Zaug et al. Nature 324:429-433 1986).Ribonuclease P contains a catalytic RNA and a small subunit protein. Itwas discovered in bacteria and is able to generate a mature 5′ end oftRNA by endonucleocatalytic cleavage of precursor transcripts(Guerrier-Takada et al. Cell 35: 849-857 1983). The mechanism ofcleavage by a hammerhead ribozyme has been characterized in the art[see, e.g., Reddy et al., U.S. Pat. No. 5,246,921; Taira et al., U.S.Pat. No. 5,500,357; Goldberg et al., U.S. Pat. No. 5,225,347].

[0055] While an understanding of the precise mechanism of the ribozymeis not necessary to practice the claimed invention, it is generallythought that the ribozyme is attached to the substrate RNA molecule byforming two paired regions via Watson-Crick pairing between the RNAsubstrate sequence and the two binding regions of the ribozyme. Thefirst deprotonation reaction takes place in the 2′ sugar at the 3′ sideof the substrate cleavage site. This deprotonation causes nucleophilicattack of the adjacent phosphodiester bond and subsequently protonationof the 5′ oxyanion cleaving group thereby generating, 2′,3′-cyclicphosphate and a 5′ hydroxyl terminus.

[0056] Like the hammerhead ribozyme, the hairpin ribozyme was also foundin plant viroids, and it acts by a similar mode of action to thehammerhead ribozyme (Feldstein et al. Gene 82:53-61 1989). The designand use of hairpin ribozymes for cleaving an RNA substrate has beendescribed in the art (Hampel et al., U.S. Pat. No. 5,527,895).

[0057] Generally, the targeted sequence of a ribozyme can vary fromapproximately 3 to 20 nucleotides long. The length of this sequence issufficient to allow a hybridization with target RNA and disassociationof the ribozyme from the cleaved RNA.

[0058] Haseloff et al (U.S. Pat. No. 6,071,730) describe trans-splicingribozymes that also provide precise cleavage sites. Trans-splicingribozymes may be used in alternative embodiments to those describedabove, in which the element of the recombinant nucleic acid thatcomprises a ribozyme is substituted therefor with a ribozyme targetsequence. The ribozyme itself is then provided in trans as a separateDNA or RNA molecule.

[0059] Thompson et al. (U.S. Pat. No. 6,183,959) describe at least sevenbasic varieties of enzymatic RNA molecules which are derived fromnaturally occurring self-cleaving RNAs. In the embodiments of theinvention which employ a ribozyme, an alternative embodiment employs theuse of a pol III promoter, since ribozymes often have extensivesecondary structure that may be more efficiently transcribed by such apromoter.

[0060] As described hereinabove, in one set of embodiments of thisinvention, one or more of the nucleic acid sequences encoding thealphavirus structural proteins is/are placed between the 5′ and 3′alphavirus replication recognition sequences, resulting in amplificationof this nucleic acid by the alphavirus replicase proteins. In preferredembodiments, a minimal 5′ alphavirus replication recognition sequence isutilized. In a specific embodiment, all of the alphavirus structuralproteins are expressed from a single promoter. The single transcript isthen resolved through a precise cleavage at the ribozyme site into tworeplicable (i.e. amplifiable) RNAs (replicable due to the presence ofthe 5′ and 3′ alphavirus replication recognition sequences), eachencoding only a subset of the alphavirus structural proteins. These RNAsare then translated from IRES sequences located 5′ to the structuralprotein coding sequences.

[0061] In a specific, preferred embodiment of the invention, theHepatitis Delta virus (HDV) ribozyme is utilized (Wu et al. Science243:652-655, 1989). The RNA of the hepatitis delta virus hasautocatalytic RNA processing activity similar to that of hammerhead andhairpin ribozymes, and the ribozyme cleavage points of both deltastrands and the domains containing them are clearly defined. The HDVribozyme is approximately 80-90 nucleotides in length and onlyfunctional in cis. Advantages in using this ribozyme are (i) there areno specific sequence requirements at the 5′ cleavage site and (ii) thecleavage product is generated with defined 3′ ends. As described in U.S.Pat. No. 5,225,337 (incorporated herein by reference), a preferredembodiment for an HDV ribozyme is a sequence consisting of at least 18consecutive nucleotides from the conserved region of HDV, in which theconserved region of the HDV RNA is found within either the region of HDVhaving ribozyme activity between residues 611 and 771 on the genomicstrand or the region between residues 845 and 980 on the complementaryanti-genomic strand. The selected sequence having ribozyme activitycleaves the target RNA molecule to form a 2′,3′ cyclic phosphate and 5′hydroxyl.

[0062] In certain embodiments of this invention, additional sequencealterations are made downstream from the ribozyme region. The ribozymecleavage event results in a clean 3′ end, such that the nucleotidesequence remains unchanged. With some ribozymes, the 5′ end of thedownstream molecule resulting from the cleavage event may containresidual ribozyme sequence, and the potential secondary structure ofthis residual sequence may have detrimental effects on downstreamtranslational activity, e.g. driven by an IRES, or upon any otherfunctional role of the 5′ sequence of the downstream fragment. Thus, tominimize the potential for such interference, in some embodiments ofthis invention it may be useful to include a region of non-translatedirrelevant nucleotide sequence downstream from the ribozyme sequence. Inan alternative embodiment to generate a clean 5′ end, a secondantigenomic ribozyme, e.g. HDV, can be added downstream from the senseribozyme. This second ribozyme would be functional only on the negativesense strand, and thus it would generate its clean “3′ end” on the 5′end of the downstream sequence.

[0063] Resolving RNA Helpers

[0064] In another embodiment of resolving helpers of this invention, thehelper construct is a DNA helper in which the promoter is aDNA-dependent RNA polymerase promoter, such as the T7 polymerasepromoter, and this promoter directs more than one alphavirus structuralprotein; preferably capsid and at least one glycoprotein. The helperfurther encodes a protease recognition sequence which is insertedin-frame between coding sequences for the alphavirus structuralproteins. Thus, disclosed is a recombinant DNA molecule for expressingalphavirus structural proteins comprising: (i) a DNA dependent RNApolymerase promoter, (ii) an IRES (iii) a nucleic acid sequence encodingat least one alphavirus structural protein, (iv) a non-autocatalyticprotease recognition site, and (v) a nucleic acid sequence encoding atleast one alphavirus structural protein, wherein the nucleic acidsequences in (iii) and (v) are not identical.

[0065] In a separate embodiment, a DNA helper for expressing a resolvingRNA helper in vivo is provided comprising (i) a DNA dependent RNApolymerase promoter, (ii) a nucleic acid sequence encoding at least onealphavirus structural protein, (iii) a non-autocatalytic proteaserecognition site, and (iv) a nucleic acid sequence encoding at least onealphavirus structural protein, wherein the nucleic acid sequences in(ii) and (iv) are not identical. In a preferred embodiment, the promoteris an RNA polymerase II promoter, such as the CMV promoter.

[0066] In another embodiment, the RNA helper is produced in vitro from arecombinant DNA molecule comprising a promoter directing thetranscription of RNA from a DNA sequence comprising (i) a first nucleicacid sequence encoding an alphavirus 5′ replication recognitionsequence, (ii) a transcriptional promoter, (iii) a nucleic acid sequenceencoding at least one alphavirus structural protein, (iv) anon-autocatalytic protease recognition site, and (v) a nucleic acidsequence encoding at least one alphavirus structural protein, and (vi)an alphavirus 3′ replication recognition sequence, wherein the nucleicacid sequences of (iii) and (v) are not identical. In preferredembodiments, the promoter is the T7 promoter and the transcriptionpromoter of element (ii) is an alphavirus subgenomic promoter.

[0067] In preferred embodiments of these helpers, the construct producesthe polyprotein Capsid-protease site-E2-E1. In other embodiments, thepolyprotein is E2-E1-protease site-Capsid or Capsid-E1-protease site-E2.Signal sequences, such as E3 or 6k, are included as appropriate, with E1and/or E2 sequences. In those constructs which encode an alphaviruscapsid protein, the capsid protein should be modified to remove theactive site for the autoproteolytic activity of capsid (see U.S. Pat.No. 5,792,462; Col 11, lines 9-19; Strauss et al. 1990 Seminars inVirology 1:347).

[0068] The protease recognition sequence is preferably a relatively raresequence that does not occur frequently in the coding sequences of thehelper cell. Examples of such proteases are widely known in the art suchas factor Xa, enteropeptidase and thrombin, but some of these proteaseshave exhibited lowered specificity for their target recognition/cleavagesites and have cleaved proteins at non-canonical sites. For this reason,the use of a rare protease with a specific cleavage recognition sitewhich would be unlikely to be present in the host cell or alphaviralprotein repertoire would be a preferred embodiment in this invention. Anexample of such a rare protease site is that of the tobacco etch virusNIa protease (TEV protease). TEV protease cleaves the amino acidsequence ENLYFQG between Q and G with high specificity. In addition,recent advances in the art have demonstrated the activity of TEVprotease can be increased significantly by a number of methods. Onemethod includes increasing the solubility of the protease by producingit in the form of a fusion protein. The second ablates the inherentauto-catalytic function of the protease which severely reduces thefunctional levels of protease within the cell. Using standardmutagenesis techniques the autocatalytic domain has been altered andleads to maintenance of high levels of the protease in its active form(Kapust et al., Protein Engineering, 14:993-1000, 2001). In the rareevent that the consensus cleavage recognition site is present within thealphaviral vector or the host cell, alternate proteases may be used inthis invention. These alternates would most likely be derived fromnon-mammalian origins to lessen the chance that the protease wouldrecognize mammalian sequences. Such proteases would preferably includeproteases derived from members of the Potyvirus family of plant virusessuch as Turnip mosaic potyvirus (TuMV) which recognizes an amino acidsequence XVRHQ where X is any aliphatic amino acid. The invention canalso utilize other proteases such as Wheat streak mosaic virus, plum poxvirus, potato virus Y, tobacco vein mottling virus, Ornithogalum mosaicvirus, yam mosaic virus, shallot potyvirus, bean yellow mosaic virus,papaya ringspot virus, pea seed-borne mosaic virus, Johnson grass mosaicvirus, rye grass mosaic virus, sweet potato mild mottle virus, or anyother members of the family of C4 unassigned peptidases. The onlyrequired feature of the protease in this invention is its restrictedability to recognize only the target sequence in the vector constructand to lack non-specific protease activity that could cleave host orother alphaviral sequences.

[0069] In the foregoing embodiments comprising a protease recognitionsite, the promoter directs the expression of all encoded alphavirusstructural proteins. The DNA helpers are introduced into the helper cellalong with a source of T7 polymerase (e.g. a stably transformedexpression cassette, a separate expression plasmid, or the polymeraseprotein), a single RNA transcript is produced from the promoter whichcontains the IRES to direct cap-independent translation of apolyprotein, which is then cleaved by the protease provided in thehelper cell. The RNA helper, transcribed from a DNA helper vector invitro, is introduced to the helper cell, preferably by electroporation,where it is amplified and translated into the polyprotein. As above, theprotease is provided to the helper cell in any one of several formats,and the polyprotein is cleaved in the presence of the protease.

[0070] In a specific embodiment, the protease may be present as a stablytransformed cassette in the genome of the helper cell. In thisembodiment, the gene encoding the protease is preferably under thecontrol of an inducible promoter, such a heat shock ormetallothionen-responsive promoter. In another embodiment, the proteasegene and/or the T7 polymerase gene can be present in the helper cell asan alphavirus subgenomic expression cassette, comprising an alphavirus5′end, an alphavirus subgenomic promoter directing expression of theprotease, and an alphavirus 3′ end. This cassette is inducible in thehelper cell, being expressed only upon introduction of the alphavirusRNA replicon to the cell. Alternatively, the protease may be added tothe helper cell concomitantly with the RNA helper and the RNA replicon,either as a separately translatable RNA or as protein.

[0071] Non-replicating DNA Helper

[0072] In another set of embodiments of this invention, a DNA helperthat does not incorporate the alphavirus replication recognitionsequences that allow amplification of the RNA encoded between thesequences is disclosed. Lacking such sequences, which can contribute tothe frequency of functional recombinant molecules that may be generatedin the helper cell, a single DNA helper encoding all the alphavirusstructural proteins necessary to produce recombinant alphavirusparticles can minimize the effect that packaging/recombination may havein a helper cell. The decrease in packaging/recombination detected, ascompared to the bipartite RNA system, is at least one order of magnitudelower; in preferred embodiments, it is two, three, or four orders ofmagnitude lower. Thus, another embodiment of the claimed inventioncomprises a recombinant DNA for expressing alphavirus structuralproteins comprising a promoter operably linked to a nucleotide sequenceencoding all the alphavirus structural proteins. In a preferredembodiment, the promoter is an RNA polymerase II promoter, such as theCMV promoter.

[0073] Preferred methods for introducing this DNA helper toalphavirus-permissive cells are also disclosed, including the use ofcationic lipids, such as FuGeneg and Lipofectamine®, electroporation, orviral vectors. The combination of cell type and transfection method canbe optimized by testing of such combinations according to the methodsdisclosed herein. In specific embodiments, 293T and Vero cells can beused. In a preferred embodiment, the DNA helper is introduced prior tothe introduction of the replicon RNA, e.g. thirty minutes, 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 12, 24, or 48 hours prior to electroporation of thereplicon RNA into the cells. Any amount of time that results in nosignificant decrease in the transfection efficiency of the replicon RNAand allows sufficient packaging of VRPs by the cells is suitable.

[0074] In an alternative embodiment, the DNA helpers of this inventionmay be co-electroporated into the helper cells with an alphavirusreplicon RNA. The parameters of electroporation are adjusted from thoseused for solely RNA or DNA electroporation to optimize the yield of VRPsfrom the helper cells.

[0075] Chimeric Alphavirus RNA Helpers

[0076] In another set of embodiments, an RNA helper which utilizesreplication recognition signals (5′ and 3′) from alphaviruses other thanthe alphavirus from which the structural proteins are derived isdisclosed. In these embodiments, the promoter directs the expression ofat least one structural protein and the alphavirus 5′ and 3′ ends thatdirect replication of the helper allow amplification, but the frequencyof packaging/recombination by these helpers is reduced, due to theinefficient or complete lack of recognition of packaging signals of theone alphavirus by the alphavirus structural proteins of the otheralphavirus. In a preferred embodiment, the RNA helper of this inventionencodes one or more VEE structural proteins and utilizes the replicationrecognition signals from an alphavirus selected from the groupconsisting of Sindbis (particularly TR339), S.A.AR86, Semliki ForestVirus, or Ross River Virus.

[0077] Non-alphavirus RNA Helpers

[0078] In another set of embodiments of this invention, helperconstructs that express one or more alphavirus structural proteins aredisclosed which utilize viral RNA replication machinery derived from avirus other than an alphavirus. Disclosed is an alphavirus structuralprotein expression system, comprising two RNA molecules, wherein (a) afirst RNA encodes sequences for viral replicase proteins, and (b) asecond recombinant RNA encodes sequences for (i) the 5′ replicationrecognition sequence for a replication complex comprising the viralreplicase proteins of (a), (ii) one or more alphavirus structuralproteins, and (iii) the 3′ replication recognition sequence for thereplication complex comprising the viral replicase proteins of (a).Following introduction of the two RNA molecules into the helper cell,the first RNA replicates the second RNA encoding one or more alphavirusstructural proteins, and then the helper cell's translational machinerytranslates the structural protein sequence(s) encoded on the secondrecombinant RNA.

[0079] In a preferred embodiment, the first and second RNA molecules arederived from a nodavirus. The Nodaviridae are a family of small,non-enveloped, isometric viruses with bipartite positive-sense RNAgenomes (Ball & Johnson, 1998 In The Insect Viruses, pp. 225-267. Editedby L. K. Miller & L. A. Ball. New York:Plenum). The nodavirus genomesare among the smallest of all known animal viruses, containing less than5 kb of genetic material. Genomic nodavirus RNA is infectious to insect,plant (Selling, B. H., et al., Proc. Natl. Acad. Sci. USA 87:434-438,1990) and mammalian (Ball, L. A., et al., J. Virol. 66:2326-2334, 1992)cells. Nodaviruses use unique regulatory cis-elements, includingnodaviral-specific RNA replication (Zhong, W., et al., Proc. Natl. Acad.Sci. USA 89:11146-11150, 1992; Ball, L. A. and Li, Y., J. Virol.67:3544-3551, 1993; Li, Y. and Ball, L. A., J. Virol. 67:3854-3860,1993; Ball, L. A., J. Virol. 69:720-727, 1995) and packaging signals(Zhong, W., et al., supra, 1992). Both genome segments are capped attheir 5′ ends but lack poly(A) tails (Newman & Brown, 1976. Journal ofGeneral Virology 30, 137-140). The smaller segment, referred to as RNA2,encodes a precursor to the nodavirus coat, or capsid, protein. Thelarger segment, referred to as RNA1, encodes the viral portion of theRNA-dependent RNA polymerase (RdRp), which replicates both RNA1 and 2(Ball & Johnson, 1998, ibid.). The RNA1 of nodaviruses are notable fortheir ability to synthesize high cytoplasmic levels of capped andfunctional mRNAs, i.e. RNA1 and RNA2. The sequences of several nodavirusRNA1 molecules have been reported recently, and the RNA2 sequences havebeen previously disclosed (see Johnson et al. 2001 J. Gen. Virol.82:1855-1866 and references cited therein, including GenBank accessionnumbers).

[0080] Applicants have determined that nodavirus replication andalphavirus replication can occur at the same time within the same cell.Thus, in one set of embodiments of this invention, a nodavirus-basedalphavirus structural protein expression system is disclosed, in whichRNA1 of a selected nodavirus is supplied to a cell in which recombinantalphavirus particles are to be packaged, along with an engineered RNA2from the same or a different nodavirus. The RNA2 is engineered to removepart of the coding sequence for the nodavirus capsid gene and substitutetherefor the coding sequence for at least one alphavirus structuralprotein. In a specific embodiment of the methods of this invention,these two nodavirus RNAs (i.e. RNA1 and engineered RNA2) areco-electroporated with an alphavirus RNA replicon into a cell in whichall three RNAs are expressed. In a preferred embodiment, a nodavirusRNA1, a recombinant nodavirus RNA2 expressing an alphavirus capsid gene,an alphavirus RNA replicon, and a second helper RNA expressingalphavirus glycoproteins are co-electroporated into a cell in which allfour RNAs can be expressed, resulting in the packaging of the repliconinto recombinant alphavirus particles. Suitable nodaviruses for use inthe claimed invention include Nodamura virus (NoV), Flock House virus(FHV), Black Beetle virus (BBV), Boolarra virus (BoV), Gypsy moth virus(GMV), and Manawatu virus (MwV). Preferred nodaviruses are FHV and NoV.In another embodiment, the RNA1 and recombinant RNA2 are introduced intothe helper cell by a virus or a virus-like particle, such as adenovirus,vaccinia, poxvirus, SV-40, adeno-associated virus, retrovirus,nodavirus, picornavirus, vesicular stomatitis virus, and baculoviruseswith mammalian pol II promoters.

[0081] Methods for Making Alphavirus Replicon Particles

[0082] Methods for making alphavirus replicon particles expressing oneor more heterologous sequences which utilize the helpers and/or thealphavirus RNA replicons of this invention are also disclosed. Using themethods of this invention, preparations of alphavirus replicon particlesare produced which contain no detectable replication-competentalphavirus particles, as determined by passage on alphavirus-permissivecells in culture.

[0083] To make the particles, a helper or combination of helpers isselected such that it provides all alphavirus structural proteinsnecessary to assemble an infectious particle. In preferred embodiments,the helper or combination of helpers encode capsid, E1 and E2. In anembodiment comprising more than one helper, one of the helpers may beselected from the helpers known in the art, e.g. the standard split RNAhelpers referenced herein. A helper or combination of helpers comprisingat least one embodiment of this invention can be used to package anyalphavirus replicon RNA. In preferred embodiments, the alphavirusreplicon RNA is a rearranged alphavirus RNA replicon as claimed hereinor the standard alphavirus replicon RNA referenced herein. In specific,preferred embodiments, the alphavirus replicon particles are VEE-basedparticles, i.e. the helpers encode VEE alphavirus structural proteinsand the replicon RNA is derived from VEE.

[0084] Alphavirus replicon particles are prepared according the methodsdisclosed herein in combination with techniques known to those skilledin the art. The methods include first introducing the selected helper(s)and an alphavirus replicon RNA encoding one or more heterologous RNAsinto a population of alphavirus-permissive cells, and then incubatingthe cells under conditions that allow for the production of alphavirusreplicon particles. The step of introducing the helper(s) and alphavirusreplicon RNA to the population of helper cells can be performed by anysuitable means, as disclosed herein or known to those generally skilledin the art. As described herein, the population of cells may be a stablytransformed cell line that provides a promoter, a trans-acting ribozyme,a protease, or any combination thereof. Alternatively, said promoters,trans-acting ribozymes or proteases may be added to the population ofcells in the form of separately relicable or non-replicable nucleicacids or proteins, as applicable.

[0085] Compositions or preparations of alphavirus replicon particles arecollected from the population of helper cells using conventionaltechniques know to those skilled in the art, e.g. U.S. Pat. Nos.5,492,462; 6,156,558; and these compositions are characterized by thefact that they will contain no detectable, replication-competentalphavirus particles, as measured by passage on alphavirus-permissivecells in culture.

[0086] As will be understood by one skilled in the art, there areseveral embodiments and elements for each aspect of the claimedinvention, and all combinations of different elements are herebyanticipated, so the specific combinations exemplified herein are not tobe construed as limitations in the scope of the invention as claimed. Ifspecific elements are removed or added to the group of elementsavailable in a combination, then the group of elements is to beconstrued as having incorporated such a change.

[0087] All references cited herein, including publications, patentapplications, and patents, are hereby incorporated by reference to thesame extent if each was individually and specifically indicated to beincorporated by reference, and was reproduced in its entirety herein.TABLE 1 PCR primers for Resolving DNA helper cloning PrimerAmplification name Sequence product GP5′CTAGCTAGCTATGTCACTAGTGACCACCATG3′ (SEQ ID NO:1) VEE glycoproteinforward GP 5′GGGCCCTCAATTATGTTTCTGGTTGGT 3′ (SEQ ID NO:2) VEEglycoprotein reverse GP-2 5′ (SEQ ID NO:3) VEE glycoprotein forwardGCAGAGCTGGTTTAGTGAACCGTATAGGCGGCGGATGAGA GAAGCGCAGACCA 3′ GP-25′GCTAGCGCTCTTCCCTTTTTTTTTTTT 3′ (SEQ ID NO:4) VEE glycoprotein reverseCapsid 5′GCTCTAGAATGTCCCGTTGCAGCCAATG 3′ (SEQ ID NO:5) VEE capsidforward Capsid 5′GCGTCGACGTCTTGGCCATAGCGGCCGCGGTTAGAGACAC (SEQ ID NO:6)VEE capsid reverse ATGGTGGTCACT 3′ hdR5′CGGGTCGGCATGGCATCTCCACCTCCTCGCGGTCCGACCT (SEQ ID NO:7) Hepatitis deltaForward GGGCATCCGAAGGAGGACGTCGTCCACTCGGATGGCTAAG ribozyme GGAGAGCTCGC 3′hdR 5′TCGAGCGAGCTCTCCCTTAGCCATCCGAGTGGACGACGTC (SEQ ID NO:8) Hepatitisdelta Reverse CTCCTTCGGATGCCCAGGTCGGACCGCGAGGAGGTGGAGAT ribozymeGCCATGCCGACCCGGGCC 3′ CMV 5′TAGTTATTAATAGTAATCAATTACGG 3′ (SEQ ID NO:9)CMV IE promoter forward CMV 5′TGGTCTGGGCTTCTCTCATGCGCCGCCTATACGGTTCACTA(SEQ ID NO:10) CMV IE promoter reverse AACCAGCTCTGC 3′ d26S5′GGCGCGCCGTCCTCCGATCGTTGTCAGAAG 3′ (SEQ ID NO:11) CMV IE + VEE 5′forward NCR d26S 5′GGCGCGCCTCCGTCAACCGCGTATACATCCTGGTAA 3′ (SEQ IDNO:12) CMV IE + VEE 5′ reverse NCR E3 5′GGCGCGCCATGTCACTAGTGACCACCATGTG3′ (SEQ ID NO:13) E3-E2-6K genes forward 6K 5′CTCGTAGGCGCCGGCGCCTGCGG 3′(SEQ ID NO:14) E3-E2-6K genes reverse EMCV5′GGCGCGGCAATTCCGCCCCTCTCCCTCCC 3′ (SEQ ID NO:15) EMCV IRES forward EMCV5′GGCGCGCCTTATCATCGTGTTTTTTCAAAG 3′ (SEQ ID NO:16) EMCV IRES reverseEMCV-2 5′GCTAGCAATTCCGCCCCTCTCCCTCCC 3′ (SEQ ID NO:17) EMCV IRES forwardEMCV-2 5′GCTAGCTTATCATCGTGTTTTTCAAAG 3′ (SEQ ID NO:18) EMCV IRES reverse

[0088] TABLE 2 PCR primers for Nodavirus RNA helper cloning PrimerAmplification name Primer sequence product Capsid F5′ CGACGCGTATGTTCCCGTTCCAGCCAATG 3′ (SEQ ID NO:19) VEE capsid MluI(MluI) Capsid R 5′GCACGCGTTTACAGACACATGGTGGTCACT (SEQ ID NO:20) VEEcapsid MluI, (MluI) 3′ VEE capsid 1, VEE capsid 2 Capsid5′CCTGCCATGGTATAAATGTTCCCGTTCCAACCA (SEQ ID NO:21) VEE capsid 1 F1 ATG3′(NcoI) Capsid 5′CCTGCCATGGCCCCGTTCCAACCAATG 3′ (SEQ ID NO:22) VEE capsid2 F2 (NcoI)

EXAMPLES

[0089] The following examples are provided to illustrate the presentinvention, and should not be construed as limiting thereof. In theseexamples, nm means nanometer, mL means milliliter, pfu/mL means plaqueforming units/milliliter, nt means nucleotide(s), PBS meansphosphate-buffered saline, VEE means Venezuelan Equine Encephalitisvirus, EMC means Encephalomyocarditis virus, BHK means baby hamsterkidney cells, GFP means green fluorescent protein, Gp meansglycoprotein, CAT means chloramphenicol acetyl transferase, IFA meansimmunofluorescence assay, and IRES means internal ribosome entry site.The expression “E2 amino acid (e.g., lys, thr, etc.) number” indicatesthe designated amino acid at the designated residue of the E2 gene, andis also used to refer to amino acids at specific residues in the E1protein and in the E3 protein.

[0090] In the Examples that follow, the starting materials forconstructing the various helper plasmids can be selected from any of thefollowing: full-length cDNA clones of VEE, i.e. pV3000, the virulentTrinidad donkey strain of VEE; or any of these clones with attenuatingmutations: pV3014 (E2 lys 209, E1 thr 272), p3042 (E1 ile 81), pV3519(E2 lys 76, E2 lys 209, E1 thr 272) and pV3526 (deletion of E3 56-59, E1ser 253), which are in the genetic background of Trinidad donkey strainVEE. As described in U.S. Pat. No. 5,792,462, these plasmids aredigested with restriction enzymes and religated to remove thenonstructural protein coding region. Alternatively, one may start withexisting helper plasmids, such as those described in Pushko et al. 1997,Ibid., or as described herein.

Example 1

[0091] VEE Replicon Particles

[0092] Replicon particles for use as a vaccine or for gene therapy canbe produced using the VEE-based vector system (see for example U.S. Pat.No. 5,792,462). In these Examples, one or more attenuating mutations(e.g. Johnston and Smith, Virology 162(2): 437-443 (1988); Davis et al.,Virology 171(1): 189-204 (1989); Davis et al. 1990) may have beeninserted into VEE sequence to generate attenuated VEE replicon particles(Davis et al., Virology 183(1): 20-31 (1991); Davis et al., Virology212(1): 102-110 (1995); Grieder et al., Virology 206(2): 994-1006(1995).

[0093] The examples herein describe the construction of an RNA replicon,i.e. an RNA that self-amplifies and expresses, and one or more helpernucleic acids encoding the structural proteins to allow packaging. Thereplicon RNA carries one or more foreign genes, e.g. a gene encoding animmunogen or a reporter gene. The replicon RNA and the helper nucleicacids (which express the alphavirus structural proteins, as describedhereinbelow) are then introduced into a single cell, i.e. the helper orpackaging cell, in which the replicon RNA is packaged into virus-likeparticles (herein referred to as “virus replicon particles” or “VRPs”)that are infectious for only one cycle. During the single, infectiouscycle, the characteristics of the alphavirus-based vector result in veryhigh levels of expression of the replicon RNA in cells to which the VRPis targeted, e.g. cells of the lymph node.

[0094] The resulting vaccine vectors are a virulent and provide completeprotection against lethal virus challenge in animals, including but notlimited to rodents, horses, nonhuman primates, and humans.

Example 2

[0095] Standard VEE Replicons and RNA Helpers

[0096] As described in U.S. Pat. No. 5,792,462, Pushko et al., 1997(Virology 239:389-401), and WO 02/03917 (Olmsted, et al.), a standardalphavirus replicon based on VEE contains the VEE nonstructural genesand a single copy of the 26S subgenomic RNA promoter followed by amultiple cloning site. In a vaccine construct, one or more genesencoding an immunogen are inserted into this cloning site. For purposesof demonstrating the capability of the novel structural proteinexpression cassettes of this invention, VEE replicons are constructed byinserting the GFP or CAT gene into this cloning site. Expression ofthese reporter genes from particles made with various combinations ofthe structural protein expression cassettes described herein demonstratethe utility and novelty of these cassettes.

[0097] The standard VEE split RNA helper systems, as described in U.S.Pat. No. 5,792,462, Pushko et al., 1997 (Virology 239:389-401), and PCTpublication WO 02/03917 (Olmsted, et al.), are described herein as “Capor Gp RNA”, “wild-type Gp RNA helper”, “glycoprotein helper”, “Gp helperRNA”, “GP-helper”, “capsid helper”, or “C-helper”. These RNA helpers aremade from DNA plasmids as described in the cited references, and theseDNA plasmids can be a convenient source for obtaining the structuralprotein coding fragments, e.g. by PCR amplification. Alternatively,these coding fragments can be obtained from full-length clones of VEE orattenuated variants thereof (see U.S. Pat. Nos. 5,185,440; 5,505,947).These standard VEE helpers are used in combination with the helperinventions disclosed herein and/or in comparative studies with the newsystems, as disclosed herein.

Example 3

[0098] Rearranged Alphavirus RNA Replicon Vector

[0099] The nsP4 region was deleted from a standard VEE GFP-expressingreplicon vector (see Example 2) by digestion with AvrII and ApaIrestriction enzymes, followed by treatment of the digested DNA with T4DNA polymerase to generate blunt ends, and re-ligation of the DNA togenerate pGFPΔnsP4-1. When RNA transcribed in vitro from the pGFPΔnsP4-1DNA plasmid is electroporated into cells, GFP is not expressed. However,GFP protein can readily be detected in cells co-electroporated withGFPΔnsP4-1 RNA and an unmodified replicon vector RNA. This demonstratesthat the pGFPΔnsP4-1 vector can be complemented by nsP4 protein providedin trans by another replicon RNA. This result indicates that the nsp4gene can function when expressed separately from the othernon-structural proteins.

[0100] The nsP4 gene (including a portion of nsP3 to maintain an nsP2protease cleavage site) was then cloned downstream of an EMCV IRES. ThensP4 region was PCR amplified from the standard VEE replicon vector withprimers nsP34-forward (SEQ ID NO: 33) and nsP4-stop (SEQ ID NO: 34)(also, see Table 3). The amplified nsP4 gene fragment was cloned into atransfer vector containing an EMCV IRES, using BamHI and XbaI as the 5′and 3′ restriction enzyme sites. A second set of primers was then usedto amplify the EMCV-nsP4 construct from the transfer vector: EMCVforward-AscI (SEQ ID NO: 35) and EMCV reverse-AscI (SEQ ID NO: 36),also, see Table 3). The EMCV-nsP4 PCR product was digested with AscIrestriction enzyme and ligated into AscI linearized pGFPΔnsP4-1 vectorDNA, to generate pGFPΔnsP4-1.1. The cloned nsP4 gene region (including aportion of nsP3 that contains the nsP2 protease site) was sequenced toensure that no mutations to the nsP4 gene were introduced duringcloning.

[0101] RNA transcribed in vitro from pGFPΔnsP4-1.1 DNA waselectroporated into Vero, BHK, 293T and CEF cells, and GFP proteinexpression was detected in all cell types. TABLE 3 PCR primers forRearranged Replicon Vector. Primer name Sequence 5′-3′ Region amplifiednsP34 CGGGATCCATGCGGTTTGATGCGGGTGCATA (SEQ ID NO:33) VEE nsP4 geneforward CATC nsP4 stop GCTCTAGATTAGCCGTAGAGAGTTATAGGGG (SEQ ID NO:34)VEE nsP4 gene EMCV TGGCGCGCCGCTCGGAATTCCCCCTCTCCC (SEQ ID NO:35)EMCV-nsP4 forward-AscI EMCV AGGCGCGCCTTCTATGTAAGCAGCTTGCC (SEQ ID NO:36)EMCV-nsP4 reverse-AscI

Example 4

[0102] Construction of Helpers with Minimal 5′ Alphavirus ReplicationRecognition Sequence

[0103] A. Constructs for Determining the Minimal 5′ Untranslated Region(UTR)

[0104] When VEE replicon particles (VRP) are inoculated onto freshcultures of Vero cells, at high multiplicities of infection (MOI),capsid protein can be detected by anti-capsid immunofluoresence assay(IFA; see Table 5 below). Detection of capsid protein in VRP infectedcells indicates that the capsid gene is present in the VRP. Similarfindings to these have been reported by others (Lu and Silver, 2001,ibid.). This may occur in at least three ways: 1) as the result of arecombination event between the capsid helper RNA and a replicon RNA, 2)as the result of copackaging of the capsid helper RNA into a VEEreplicon RNA containing particle, or 3) as a result of packaging thecapsid helper RNA alone into particles (no VEE replicon RNA present).VEE helpers previously described in the art (Johnston et al., ibid.,Pushko et al., ibid.) contain 519 nucleotides of the 5′ region of VEERNA. Specifically, this 5′ region encodes a 45 nt untranslated region(UTR) as well as 474 nt of the nsP1 open reading frame (ORF). Thesehelper RNAs were originally designed to remove the VEE nucleotide regionthought to be involved in packaging of viral RNAs into particles. Thisdesign was based on work carried out using Sindbis virus, in which aregion of nsP1, located approximately 1000 nt into the genome, wasthought to encode the packaging signal (Bredenbeek, P J et al., 1993 JVirol 67: 6439-6446; Levis et al 1986 Cell 44:137-145; Weiss et al. 1989J Virol 63:5310-8). To further optimize the helper constructs,increasing deletions were made into the 5′ UTR to determine the minimalsequences required for the helpers to provide (i) acceptable VRP yieldsand (ii) the lowest theoretical frequency for capsid genecopackaging/recombination in VRP preparations.

[0105] 1. Construction of 5′ Truncated VEE Capsid Helpers

[0106] A capsid helper plasmid containing the sequence for the VEEcapsid gene was constructed as previously described. This constructcontains a sequence of 519 nucleotides, the “5′ UTR”, located upstream(i.e. 5′) from the ATG initiation codon for the capsid coding sequence,and is herein referred to as “hcap10”.

[0107] (a) PCR-based Construction

[0108] Nine consecutive deletions of approximately 50 nt each were madein the 519 nt UTR present in the VEE capsid helper (see Example 2). Thefollowing deletions were made from the 3′ end of the 5′ UTR: HcapConstruct name: 5′ UTR nt included: 10 1-520 1 1-484 2 1-435 3 1-397 41-336 5 1-294 6 1-231 7 1-185 8 1-125 9 1-46 

[0109] This initial set of capsid helper (“Hcap”) constructs wasproduced using a PCR method that did not require cloning of individualconstructs. The procedure was carried out in two separate steps. First,nine reverse primers (Hcap 1-9 reverse) were designed ˜50 nt apart,complementary to the 5′ UTR up to position 470 of the VEE replicon, andengineered to contain an ApaI restriction site: Primer name SEQ ID NO:48-132.pr2 39 48-132.pr4 40 48-132.pr6 41 48-132.pr8 42 48-132.pr10 4348-132.pr12 44 48-132.pr14 45 48-132.pr16 46 48-132.pr18 47

[0110] The sequences of each of these primers is presented in Table 4.An Hcap forward primer, 13-101.pr1 (SEQ ID NO: 37) (also, see Table 4)was designed so that when it was used in combination with any one of thereverse primers, it would amplify a fragment containing the T7 promoterand the respective 5′UTR deletion. Second, primers were designed toamplify the capsid gene out of the existing capsid helper plasmid suchthat the fragment would have the following composition: 5′ ApaIrestriction site, 26S promoter, capsid gene, 3′ UTR-3′. These primersare 48-132.pr1 (SEQ ID NO: 48) and 3-8pr4 (SEQ ID NO: 49) (also, seeTable 4). The amplified PCR products were digested with ApaI enzyme andligated together at the common ApaI site. These ligated DNAs were usedas template to PCR amplify each of the Hcap constructs using primers13-101.pr1 (SEQ ID NO: 37) and 13-101.pr4 (SEQ ID NO: 38), which flankthe 5′ and 3′ regions of each helper, respectively. The amplified Hcaphelper DNAs were used to transcribe RNA in vitro for use in VRPpackaging experiments. TABLE 4 Primers for Hcap construct cloning.Primer name Primer sequence 5′-3′ Region amplified 13-101.prlCCGGGAAAACAGCATTCCAGGTATTAGA (SEQ ID NO:37) Heap forward 13-101.pr4TTTTTTTTTTTTTTTTTTTTTTTTTTTT (SEQ ID NO:38) VEE 3′ reverseTTTTTTTTTGAAATATTAAAAAACAAAATCCGAT TCGG 48-132.pr2GACGGGCCCCTTGCCCTTCGTAGCGACAC (SEQ ID NO:39) Heap-1 reverse 48-132.pr4GACGGGCCCAGTTTCCAGGTCAGGGTCGC (SEQ ID NO:40) Hcap-2 reverse 48-132.pr6GACGGGCCCCCTTCATTTTCTTGTCCAATTCCT (SEQ ID NO:41) Hcap-3 reverse48-132.pr8 GACGGGCCCTGCATACTTATACAATCTGTCCG (SEQ ID NO:42) Hcap-4reverse GA 48- GACGGGCCCCGGACAGATACAATGATACTTGT (SEQ ID NO:43) Hcap-5reverse 132.pr10 GCT 48- GACGGGCCCGCCAGATGCGAAAACGCTCTG (SEQ ID NO:44)Hcap-6 reverse 132.pr12 48- GACGGGCCCGCCAGATGCGAAAACGCTCTG (SEQ IDNO:45) Hcap-7 reverse 132.pr14 48- GACGGGCCCTACCTCAAACTGCGGGAAGC (SEQ IDNO:46) Hcap-8 reverse 132.pr16 48- GACGGGCCCTTTTGGGTAGGTAATFFGGTCTGG(SEQ ID NO:47) Hcap-9 reverse 132.pr18 48-132.pr1GACGGGCCCCTATAACTCTCTAC (SEQ ID NO:48) Capsid forward 3-8pr4GCAACGCGGGGAGGCAGACA (SEQ ID NO:49) Capsid reverse

[0111] (b) Plasmid Construction of Selected Truncated Helpers

[0112] To construct plasmid versions of selected Hcap clones, each 5′UTR was PCR amplified as described above, digested with EcoRI and ApaIrestriction enzymes, and then ligated into a standard VEE repliconvector (see Example 2), which was also digested with EcoRI and ApaIrestriction enzymes. The resulting “empty” helper vectors (Δ-helpers)each contained one of the nine deleted 5′ UTR regions. The capsid genewas then PCR amplified from a standard capsid helper plasmid withprimers 48-132.pr1 and 3-8pr4, as described above, digested with ApaIand NotI restriction enzymes and ligated into each of the Δ-helpers alsolinearized with ApaI and NotI enzymes.

[0113] B. Analysis of Truncated Helper Constructs

[0114] Each of the truncated helper constructs was tested separately forits ability to package replicon RNA expressing an immunogen (e.g. theGag protein from HIV) into VRPs. 30 ug of each of the 3 RNAs (i.e. thetruncated capsid helper, standard VEE Gp helper, and anHIV-Gag-expressing replicon RNA) were electroporated into CHO or VEROcells.

[0115] 1. IFA Analysis of Electroporated CHO Cells TABLE 5 IFA Analysisof Electroporated CHO Cells Sample GAG capsid Gp Hcap1 >90% 20% >90%Hcap2 >90% 15% >90% Hcap3 >90% 15% >90% Hcap4 >90% 10% >90% Hcap5 >90%5% >90% Hcap6 >90% 5% >90% Hcap7 >90% 5% >90% Hcap8 >90% <1% >90%Hcap9 >90% <1% >90% Hcap10 >90% >90% >90%

[0116] 2. Packaging/recombination Analysis in CHO Cells

[0117] Packaging/recombination studies were carried out on VRP generatedin CHO cells (Table 6). The titer of the resultant GAG VRP and capsidexpressing particles was determined by infection of fresh cells andperforming IFA for GAG or capsid protein. High titered VRP (>1×10^ 8/ml)were generated in CHO cells using Hcap-1 through Hcap-6. Most CHO cellVRP preparations generated with these Hcap RNAs had 20 to >100 foldreduction in capsid copackaging/recombination titer, as compared withHcap10 (the standard, “full length 5′UTR” capsid helper). TABLE 6 GAGVRP capsid fold reduction Sample Titer/ml titer/ml capsid titer vs hcap10 Hcap1 2.5 × 10⁸ 1.4 × 10³ 23.6 Hcap2 1.0 × 10⁸ 1.4 × 10³ 23.6 Hcap31.8 × 10⁸ 7.1 × 10² 46.5 Hcap4 1.4 × 10⁸ 2.6 × 10² 126.9 Hcap5 1.1 × 10⁸1.4 × 10³ 23.6 Hcap6 1.7 × 10⁸ 6.7 × 10³ 4.9 Hcap7 8.8 × 10⁷ 1.4 × 10³23.6 Hcap8 2.8 × 10⁶ nd nd Hcap9 7.4 × 10⁶ 3.5 × 10² 94.3 Hcap10 3.4 ×10⁸ 3.3 × 10⁴ 0.0

[0118] 3. Packaging/recombination Analysis in Vero Cells Using SelectedHelpers

[0119] Plasmid clones were prepared for the truncated 5′ UTRs of Hcap1through Hcap4, and the clones were sequenced to confirm their identity.Using the Hcap4 truncated helper to provide the capsid protein, VRP wereproduced following co-electroporation into Vero cells with an HIV-GagVEE replicon RNA and a standard Gp RNA helper (see Example 2).

[0120] IFA analysis of Vero cells infected with these VRP at an MOI of˜50-100 (to ensure 100% infection of cells) was carried out 16 hr postinfection using an anti-capsid antibody. The number of cells that wereIFA-positive for capsid was determined per field at a 10× magnification,and this number was used to calculate a copackaging-recombination titerfor capsid on a per milliliter basis. Table 7 provides the results usingthis truncated helper, as compared with the standard capsid helper(“Hcap 10”). TABLE 7 VRP and Capsid titers in Vero cells using Hcap4Helper GAG VRP capsid fold reduction Sample Titer/ml titer/ml capsidtiter vs hcap10 Hcap4 9.6 × 10⁸ 1.3 × 10⁴ 36.2 Hcap10 1.2 × 10⁹ 4.7 ×10⁵ 0.0

Example 4

[0121] T7 Polymerase-driven Helpers

[0122] A. T7 Polymerase-driven VEE Glycoprotein Helper

[0123] The glycoprotein (Gp) genes from the Gp helper are amplified byPCR and cloned downstream from an EMC IRES. The EMC/Gp fragment is thencloned into pCRBlunt (Invitrogen, Carlsbad, Calif.) under the control ofa T7 RNA polymerase promoter. Transfection of this pCRBlunt-EMC/Gp DNA(Fugene 6, Bochringer Mannheim, Indianapolis, Ind.) into BHK-21 cellsexpressing T7 RNA polymerase protein resulted in VEE Gp proteinexpression in cells transfected with the DNA, as determined by IFA.

[0124] B. Resolving T7-polymerase Driven VEE DNA Helper

[0125] Construction of VEE DNA Resolving Helper

[0126] The VEE capsid protein is amplified from standard C helperplasmid (see Example 2) using the VEECapF/XbaI primer (SEQ ID NO: 23)and the VEECapR/BamHI primer (SEQ ID NO: 24) (also, see Table 8). Theresulting PCR product is cloned into pCR4-TOPO (InVitrogen Corp.,Carlsbad, Calif.), resulting in pC4-Vcapsid, and orientation is verifiedby restriction enzyme analysis with SpeI. The VEE glycoprotein genes arePCR amplified from the standard Gp helper plasmid using theVEEGpTEP/BamHI primer (containing the 21 nt sequence encoding thetobacco etch virus protease recognition sequence (TEP); SEQ ID NO: 25)and the VEEGpR/EcoRV primer (SEQ ID NO: 26) (also, see Table 8) followedby restriction enzyme digest with BamHI. The pCR4-VCapsid clone isdigested with BamHI and PmeI and ligated with the Bam HI-digested VEEGpPCR product to generate a structural protein coding cassette with theprotease recognition sequence in between the capsid and Gp sequences.The EMCV IRES is PCR amplified from pIRES using EMCV-2 forward (SEQ IDNO: 17) and reverse (SEQ ID NO: 18) primers, digested with NheI andcloned into the XbaI restriction enzyme site of pCR4-VSp, andorientation is verified by PCR analysis with EMCV-2 forward andVEECapR/BamHI. TABLE 8 Primers for T7 promoter driven DNA ResolvingHelper Primer Primer Sequence VEECapF/XbaI5′-GTCTAGAATGTTCCCGTTCCAACCAATG-3′ (SEQ ID NO:23) VEECapR/Bam5′-CGGGATCCCCATTGCTCGCAGTTCTCCGG-3′ (SEQ ID NO:24) VEEGpTEP/Ba5′-CGGGATCCGAAAACCTGTATTTTCAGGGCATGTCACT (SEQ ID NO:25) mHIAGTGACGACCATGTGTCTGCTCGCC-3′ VEEGpR/EcoR5′-CGATATCTCAATTATGTTTCTGGTTG-3′ (SEQ ID NO:26) V

Example 5

[0127] Pol II Promoter-driven Non-replicating Helpers

[0128] A. Construction of Helpers

[0129] Alphavirus structural protein genes are PCR amplified using genespecific primers that possess unique restriction enzyme sites and cloneddownstream of a DNA Polymerase II promoter for gene expression intransfected cells. In the case of VEE constructs, capsid and/or Gp geneswere PCR amplified from the C-helper or Gp-helper and cloned intopCDNA-3.1 (In Vitrogen, Carlsbad, Calif.) under the control of a CMVpromoter. None of the clones retained any VEE 5′UTR, 3′UTR or 26S RNAsequences. The following constructs were generated: Name: ExpressionProduct: pCDNA-VCap VEE capsid protein pCDNA-VcapCleave VEE capsidprotein and 8 amino acids of the E3 protein pCDNA-VGp(X/X/N) VEE GpspCDNA-VSp VEE capsid protein and Gps (all VEE structural proteins)

[0130] To construct pCDNA-VSp, the VEE glycoprotein gene was PCRamplified from the standard Gp helper plasmid (see Example 2) using theVEEGpF/XbaI primer (SEQ ID NO: 27) and the VEEGpR/NheI primer (SEQ IDNO: 28) (also, see Table 9). The resulting PCR product was digested withXbaI and NheI restriction enzymes and cloned into the XbaI site ofpCDNA3.1 (−) to generate pCDNA-VGp(X/N). Orientation with respect to theCMV immediate early promoter was verified by restriction enzyme analysiswith XbaI and SpeI. The full-length VEE capsid gene was PCR amplifiedfrom the standard C helper plasmid (see Example 2) using theVEECapF/XbaI primer (SEQ ID NO: 29) and the 3-42pr4 primers (SEQ ID NO:30) (also, see Table 9). This PCR product was digested with XbaI andligated into pCDNA-VGp(X/N) at the XbaI restriction enzyme site togenerate pCDNA-VSp. Orientation was verified by restriction enzymeanalysis with SpeI. TABLE 9 Primers for constructing pCDNA-VSp PrimerSequence VEEGpF/XbaI 5′-GTCTAGAATGTCCCTAGTGACCACCATG-3′ (SEQ ID NO:27)VEEGpR/NheI 5′-GCGCTAGCGTCAATTATGTTTCTGGTTG-3′ (SEQ ID NO:28)VEECapF/XbaI 5′-GTCTAGAATGTTCCCGTTCCAACCAATG-3′ (SEQ ID NO:29) 3-42pr45′-CAATCGCCGCGAGTTCTATG-3′ (SEQ ID NO:30)

[0131] B. Transfection with pCDNA-VSp

[0132] 293T cells in T-175 flasks were transfected with 20 μg pCDNA-VSpDNA using Fugene® 6 (Roche, Indianapolis, Ind.) or Lipofectamine® 2000(LF2K; Gibco-BRL, Gaithersburg, Md.) and cells were incubated for 6 or24 h at 37° C. with 5% CO₂. Cells were trypsinized, washed twice in PBSand resuspended at 1.2×10⁷ cells/ml in 800 μl PBS. Cells were then mixedwith 30 μg of an alphavirus replicon RNA encoding GFP and transferred toa 0.4 cm gap electroporation cuvette. The cells and RNA were pulsedthree times at 450 V and 25 μF. After 10 minutes at room temperature,the cells were seeded into T75 flasks with 25 mls DMEM+10% FBS. Samplesof each electroporation were aliquoted to 96 well plates forimmunofluorescence analysis and all cells were incubated at 37° C. with5% CO₂.

[0133] At approximately 16 h post-electroporation, cells in 96 wellplates were fixed with 2% formaldehyde; 0.2% gluteraldehyde and analyzedfor GFP protein expression. Samples were alternatively fixed with MeOHand analyzed by IFA for VEE capsid and Gp protein expression. Resultsare summarized in Table 10, as a percentage of the electroporated cellsexpressing protein. TABLE 10 Percentage of cells expressing protein 16 hafter electroporation. VEE VEE Helper Time* GFP Capsid Gp Cap/Gp  6 h50% 50% 50% RNA Cap/Gp 24 h 80% >50% >50% RNA VSp  6 h 80% <1% 1%(Fugene) VSp 24 h >80% <1% 30% (Fugene) VSp  6 h 80% 10% 50% (LF2K) VSp24 h >80% 1% 80% (LF2K)

[0134] Western analysis demonstrates the presence and proper processingof the capsid protein in cells transformed with pCDNA-VSp.

[0135] C. GFP-expressing VRP Production Using DNA Helpers

[0136] Each of the constructs described in (A.) were tested separatelyor in combination for their ability to package replicon particlesexpressing a reporter (e.g. GFP protein).

[0137] 1. 293T cells

[0138] 293T cells transformed with DNA constructs were electroporatedwith 30 ug GFP-expressing RNA. 30 ug of Capsid or Gp helper RNAs wereincluded in electroporation of cells receiving only Gp or Capsid DNAsrespectively. At approximately 24 h after replicon RNA electroporationas described in (B.), the culture medium was harvested and cell debriswas removed by centrifugation in a swinging bucket rotor for 5 min. at2000 rpm. The supernatant was transferred to new 50 ml conical tubes andstored at 4° C.. Ten-fold serial dilutions of clarified culture medium,containing alphavirus GFP-VRPs, were prepared, and 30 μl of eachdilution were inoculated onto Vero cells in 96-well plates. Cells wereincubated overnight at 37° C. with 5% CO₂ followed by fixation in 2%formaldehyde/0.2% glutaraldehyde. Titrations were determined by countingthe number of cells that were GFP positive in 5 fields at the lowestpossible dilution. Results are summarized in Table 11 below: TABLE 11GFP VRP Production using DNA Helpers Titer/ml IFA for IFA for IFA forSample VRP GFP* Capsid* Gp* Control** 1.5 × 10⁶ >80% 20% 20% VCap 6.4 ×10⁴ 50% 20% 10% CapCleave 1.0 × 10⁵ 50% 20% 10% VGp(X/X/N) 4.3 × 10⁴ 50%20% 20% VSp 1.9 × 10⁵ 50% 1% 30% CapCleave + 4.3 × 10⁴ 50% 20% 20%VGp(X/X/N)

[0139] 2. Optimization of DNA Transfection in 293T Cells Followed byElectroporation Using pCDNA-VSp

[0140] At approximately 24 h after replicon RNA electroporation asdescribed in (B.), the culture medium was harvested and cell debris wasremoved by centrifugation in a swinging bucket rotor for 5 min. at 2000rpm. The supernatant was transferred to new 50 ml conical tubes andstored at 4° C. Ten-fold serial dilutions of clarified culture medium,containing alphavirus GFP-VRPs, were prepared, and 30 μl of eachdilution were inoculated onto Vero cells in 96-well plates. Cells wereincubated overnight at 37° C. with 5% CO₂ followed by fixation in 2%formaldehyde/0.2% glutaraldehyde. Titrations were determined by countingthe number of cells that were GFP positive in 5 fields at the lowestpossible dilution. Results are summarized in Table 12. TABLE 12 VRPtiters generated using pCDNA-VSp helper Helper Time* Titer (VRP/ml)Cap/Gp RNA  6 h 1.49 × 10⁷ Cap/Gp RNA 24 h 5.69 × 10⁷ VSp (Fugene)  6 h2.13 × 10⁵ VSp (Fugene) 24 h 2.13 × 10⁶ VSp (LF2K)  6 h 9.26 × 10⁵ VSp(LF2K) 24 h 2.85 × 10⁶

[0141] D. HIV-Gag Expressing VRP Production Using pCDNA-VSp Helper

[0142] 293T cells in T175 flasks were transfected with pCDNA-VSp DNA, asabove, collected 6 h later, and then electroporated with 30 μg HIV-Gagreplicon RNA (see Olmsted, et al., WO 02/03917). Electroporated cellswere seeded into T75 flasks and samples were aliquotted into 96-wellplates. At 16 h post-electroporation, samples of the helper cells in96-well plates were fixed in methanol and analyzed by IFA for HIV-Gag,VEE Capsid or VEE Gp protein expression.

[0143] Culture medium containing the VRPs released from electroporatedcells was collected, and titrations to determine recombinant alphavirusparticle yield were measured. Titers for HIV-Gag expressing VRP, as wellas Capsid and Gp copackaging/single recombinants, were determined by IFA(Table 13). TABLE 13 GAG VRP titers and copackaging with VSp DNAhelpers. HIV-Gag Helper (VRP/ml) Capsid (FFU/ml) Gp (FFU/ml) Cap/Gp RNA7.75 × 10⁶ 1.03 × 10³ 1.42 × 10³ pCDNA-VSp 1.64 × 10⁶ none detected nonedetected

[0144] The results in Table 13 demonstrate that the HIV-Gag VRP titersfrom the pCDNA-VSp helper were less then 10-fold lower than titersobtained with the bipartite RNA helper system (“Cap/Gp RNA”) while thepackaging/recombination frequencies were decreased by at least threeorders of magnitude.

Example 6

[0145] Construction of Resolving DNA Helpers

[0146] A. Resolving DNA Helper A

[0147] To construct this embodiment of the invention, alphavirus capsidand glycoprotein structural protein genes are cloned into two separatepositions in the single helper DNA molecule. At least one structuralgene, located in the first position, is cloned directly downstream froma DNA dependant RNA polymerase II (po II) promoter. One or morestructural genes, not encoded in the first position, are located in thesecond position, being positioned downstream of the first position, suchthat the transcript resulting from the pol II promoter-directedexpression contains an IRES element directly 5′ to the structuralprotein gene(s) in the downstream position.

[0148] One method of construction employs first PCR amplifying theselected alphavirus capsid or glycoprotein structural proteins usingstructural-gene specific primers that also code for unique restrictionenzyme sites. The sequence encoding the structural protein gene(s)located in the first position is cloned directly into the DNA dependentRNA polymerase II (pol II) promoter-based expression vector of choice.The sequence encoding the structural protein gene(s) located in thesecond position is initially cloned into a transfer vector that containsan IRES sequence such that the transcript eventually resulting from thepromoter-directed expression will contain an IRES element directly 5′ tothe structural protein gene(s) coding sequence in the second position.

[0149] To insert the ribozyme sequence, overlapping complementaryprimers that encode a ribozyme are annealed to produce a ribozyme linkersequence with unique 5′ and 3′ restriction sites at each end. Then, theIRES-structural protein gene fragment is digested out of the transfervector, and the pol II expression vector is digested at the unique 3′restriction site of the sequence encoding the gene(s) in the firstposition and at another unique restriction site further downstream thatis compatible with the 3′ site of the IRES-structural protein gene DNAfragment. The construct is completed by ligating the IRES-structuralprotein gene fragment and the po II expression vector together at thecompatible 5′ and 3′ sites at the ends of the ribozyme linker sequence.

[0150] Construction of a VEE Helper A

[0151] The VEE glycoprotein (GP) gene is amplified with GP forward (SEQID NO: 1) and GP reverse primers (SEQ ID NO: 2) (also, see Table 1) andthe VEE capsid (C) gene is amplified with C forward (SEQ ID NO: 5) and Creverse primers (SEQ ID NO: 6) (also, see Table 1) using GP-helper andC-helper plasmids as templates for PCR, respectively (e.g. Pushko et al,1997). The VEE GP PCR product is digested with NheI and ApaI restrictionenzymes and ligated into a suitable vector linearized with the samerestriction enzymes and containing a polII promoter, such as thecommercially available pCDNA3.1 (Invitrogen, Carlsbad, Calif.; note thatthis vector, as well as other commercially available vectors, isengineered with a selectable marker for use of the purchased plasmid inmammalian cell culture, but since the claimed inventions do not requiresuch a marker, it is removed prior to insertion of the VEE structuralprotein coding sequence(s)). In employing pCDNA3.1, the VEE GP gene isthen located downstream of the CMV immediate early (IE) promoter,generating pCDNA3.1 /sp1.

[0152] The VEE capsid PCR fragment is digested with XbaI and SalIrestriction enzymes and ligated into a suitable vector linearized withthe same restriction enzymes and containing as IRES, such as thecommercially available XbaI/SalI linearized pIRES DNA (Clontech, PaloAlto, Calif.). This manipulation generates pIRES/sp2. Overlappingcomplementary primers hdR-Forward (SEQ ID NO: 7) and hdR-Reverse (SEQ IDNO: 8) (also, see Table 1), coding for the hepatitis delta ribozyme(hdR), are annealed together to generate an hdR linker sequence withApaI and XhoI restriction sites at the 5′ and 3′ ends, respectively. Thevector portion of the construct is produced by digesting pCDNA3 1/sp1with ApaI and NotI restriction enzymes. A 1502 base pair XhoI/NotIIRES/sp2 fragment digested from pIRES/sp2 DNA and the ApaII/XhoI hdRlinker fragment are then ligated with ApaI/NotI linearized pCDNA3.1/sp1to generate VEE Helper A.

[0153] B. Resolving DNA Helpers B and C

[0154] To construct other embodiments of the invention, an alphavirusstructural protein helper vector (e.g. comprising an alphaviral 5′replication recognition sequence, either an alphavirus transcriptionalpromoter (herein Helper B; e.g. an 26S alphavirus subgenomic promoter)or an internal ribosome entry site (IRES) (herein Helper C; e.g. theEMCV IRES), a nucleic acid sequence encoding the upstream alphavirusstructural protein gene(s), and an alphavirus 3′ replication recognitionsequence) is cloned directly into a polII promoter-based expressionvector of choice (see Example 5). Concomitantly, the nucleic acidsequence encoding the alphavirus structural protein gene(s) in thedownstream fragment is cloned into a transfer vector containing an IRESsequence, as described herein. To assemble Helper B, the IRES-structuralprotein coding sequence fragment is then digested out of the transfervector and the released fragment is ligated into the promoter expressionvector, so that the polII promoter directs the expression of thestructural proteins in both the upstream and downstream positions.

[0155] Helper C is prepared in two steps from Helper B. The first stepis to remove the 26S promoter from Helper B, while introducing a uniquerestriction site in its place. The second step is to clone an IRESsequence into the unique engineered restriction site.

[0156] Construction of a VEE Helper B.

[0157] The CMV IE promoter is PCR amplified from pIRES2-DsRed2(Clontech) using the CMV forward primer (SEQ ID NO: 9) and the CMVreverse primer (SEQ ID NO: 10) (also, see Table 1). The VEE 5′non-coding region, 26S promoter, GP gene and VEE 3′ noncoding region isamplified from a GP-helper plasmid (see Example 4A) using the GP-2forward primer (SEQ ID NO: 3) and the GP-2 reverse primer (SEQ ID NO: 4)(also, see Table 1). The CMV IE PCR product and the GP-helper PCRproduct have a 53 nt region of homology at their 3′ and 5′ends,respectively. This region of homology allows an overlapping PCR reactionto produce a gene construct containing the CMV IE promoter thatinitiates transcription at the VEE 5′ end of the GP-helper sequence. TheCMV IE/GP-helper fragment is then digested with NheI restriction enzyme.The pol II promoter-based vector pCDNA3.1 is linearized with BglIIrestriction enzyme and then treated with T4 DNA polymerase to produce ablunt end. The vector is further digested with NheI to release theexisting CMV IE and T7 RNA polymerase promoters from the vector. TheNheI digested CMV IE/GP-helper PCR product is then ligated into theBglII(T4 treated)/NheI digested pCDNA3.1 vector, generatingpCDNA3.1/sp1.2. The 1502 base pair XhoI/NotI IRES/sp2 fragment digestedfrom pIRES/sp2 DNA and the ApaI/XhoI hdR linker fragment are thenligated with ApaI/NotI linearized pCDNA3.1/sp1.2 to generate VEE HelperB.

[0158] Construction of VEE Helper C.

[0159] Removal of the 26S promoter in Helper B is accomplished by PCR,using two sets of primers. The first set of primers (d26S forward (SEQID NO: 11) and d26S reverse (SEQ ID NO: 12); also, see Table 1) amplifya fragment containing the CMV IE and the VEE 5′ non-coding region (NCR).The d26S reverse primer anneals upstream from the 26S promoter so thatit is not included in the PCR product. The CMV IE/VEE 5′ NCR PCR productencodes a 5′ PvuI site, found in the backbone of Helper B DNA, and anengineered unique AscI restriction site at the 3′ end of the VEE 5′ NCR.The second set of primers (E3 forward (SEQ ID NO: 13) and 6K reverse(SEQ ID NO: 14); also, see Table 1) amplify a product containing the VEEE3-6K region of the GP-helper that also does not contain the 26Spromoter. The E3-6K product contains an engineered 5′ AscI site and a 3′unique SgrAI restriction site found in the 6K gene. After PCRamplification, the CMV IE/VEE 5′ NCR PCR product is digested with PvuIand AscI restriction enzymes, and the E3-6K product is digested withAscI and SgrAI restriction enzymes. Helper B DNA (see above) is digestedwith PvuI and SgrAI restriction enzymes to release the CMV IE-VEE 5′NCR-26S-E3-6K region. A new Helper is then reconstituted by ligating thePvuI/AscI CMV IE/VEE 5′ NCR digested fragment and the AscI/SgrAI E3-6Kdigested fragment with the PvuI/SgrAI digested Helper B vector,generating Helper B.2. Helper B.2 is identical to Helper B except thatthe 26S promoter has been removed and a unique AscI restriction site hasreplaced it.

[0160] The EMCV IRES is amplified from the pIRES vector using the EMCVforward primer (SEQ ID NO: 15) and the EMCV reverse primer (SEQ ID NO:16) which both contain engineered 5′ AscI restriction sites (also, seeTable 1). After amplification, the EMCV IRES PCR product is digestedwith AscI restriction enzyme and ligated into AscI linearized Helper B.2DNA, generating Helper C.

[0161] C. Resolving DNA Helper D

[0162] Another embodiment of this invention can be constructed bymodifying Helper A to contain an IRES element that directscap-independent translation of the alphavirus structural proteins in theupstream as well as the downstream location, a construct referred toherein as Helper D.

[0163] Construction of a VEE Helper D.

[0164] The EMCV IRES is amplified from the pIRES vector using the EMCV-2forward primer (SEQ ID NO: 17) and the EMCV-2 reverse primer (SEQ ID NO:18), each containing engineered 5′ NheI restriction sites (also,seeTable 1). After amplification, the EMCV IRES PCR product is digestedwith NheI restriction enzyme and ligated into NheI linearized VEE HelperA, generating VEE Helper D.

Example 7

[0165] Chimeric Alphavirus RNA Helpers

[0166] A. Construction of VEE-SIN Helper

[0167] The VEE capsid gene was cloned into the XbaI restriction site ofthe pSINrep5 replicon vector (InVitrogen, Carlsbad, Calif.).Sindbis-based helpers that contain the VEE capsid gene were constructedby deleting portions of the Sindbis nsP region from thepSINrep5/VEEcapsid construct. The pΔSIN/VEEcapsid DNA was prepared bydigesting pSINrep5/VEEcapsid DNA with SmaI and BamHI restrictionenzymes, deleting 6567 base pairs (bp) of the nsP genes. A second helperconstruct (pΔSIN/VEEcap-2), lacking an additional 667 bp of the nsPregion, was prepared by PCR amplifying a region of pΔSIN/VEEcapsid,using the primers ΔSIN26S/RsrII forward (SEQ ID NO: 31) andΔSIN-ApaI/reverse (SEQ ID NO: 32) (also, see Table 14) and ligating itinto RsrII and ApaI linearized pΔSIN/VEEcapsid DNA. TABLE 14 PCR primersused to generate ASIN/VEEcap-2 Region primer Sequence 5′-3′ amplifiedΔSIN26S/RsrII TTTCGGACCGTCTCTACGGTGGTCCTAAAT (SEQ ID NO:31) nsP and VEEforward AGTC capsid ΔSIN-Apal/ CTGGTCGGATCATGGGCCC (SEQ ID NO:32) nsPand VEE reverse capsid

[0168] B. HIV-Gag Expressing VRP Production Using a Chimeric AlphavirusHelper

[0169] The ΔSIN/VEEcapsid or ΔSIN/VEEcap-2 helpers were used to generateVRP in Vero cells with a VEE Gp RNA helper (as described herein) and aVEE RNA replicon expressing the HIV Gag gene. The culture mediumcontaining HIV-Gag VRP, generated using either the ΔSIN/VEEcapsid orΔSIN/VEEcap-2 helper, was collected, and the yield of VRPs wasdetermined using an HIV-Gag IFA. Using the ΔSIN/VEEcapsid helper, a VRPtiter of 2.6×10⁶/ml was obtained; using the ΔSIN/VEEcap-2 helper, a VRPtiter of 1.9×10⁶/ml was obtained.

Example 8

[0170] Construction of Nodavirus-based Helper RNAs

[0171] Generally, the constructs are produced as follows. The alphaviruscapsid gene is PCR amplified with gene specific primers that introducerestriction enzyme sites. The PCR amplified product is cloned directlyinto a Nodavirus RNA2 (generated from either flock house virus (FHV) ornodamura (NoV)), which are digested with compatible restriction enzymes.

[0172] Construction of Nodavirus VEE Capsid Helpers

[0173] The VEE capsid gene is PCR amplified with the capsid F (MluI)primer (SEQ ID NO: 19) and the capsid R (MluI) primer (SEQ ID NO: 20)that introduce a MluI site, (also, see Table 1), using pCDNA VSp (seeExample 5) as the template. The VEE capsid PCR product is digested withMluI and ligated into the MluI digested FHV RNA2 vector and the BssHIIdigested NoV RNA2. BssHII digestion produces compatible cohesive endswith MluI. The resultant clones are sequenced to determine orientationand to verify sequence accuracy.

[0174] Alternatively, the VEE capsid gene can be directionally clonedinto the NcoI and MluI sites of FHV RNA2 and the NcoI and BssHII sitesof NoV RNA2. This NcoI site is 5′ to the MluI or BssHII sites in the FHVand NoV RNA2, respectively, separated by one nucleotide. The VEE capsidgene is PCR amplified with one of two forward primers containing an NcoIsite, capsid F1 (NcoI) (SEQ ID NO: 21) and capsid F2 (NcoI) (SEQ ID NO:22), and a reverse primer that contains a MluI site, capsid R (MluI)(SEQ ID NO: 20), using pCDNA VSp (see Example 5) as the template. Theresultant VEE capsid 1 and VEE capsid 2 PCR products are digested withNcoI and MluI and ligated into the NcoI/MluI digested FHV RNA2 vectorand the NcoI/BssHII digested NoV RNA2 vector.

[0175] In addition, the VEE glycoprotein gene and the entire VEEstructural protein gene cassette can be cloned into the FHV RNA2 and theNoV RNA2 expression vectors as described above using appropriateprimers.

[0176] The nodavirus capsid helper described herein is co-electroporatedinto VERO cells at 28 C with nodavirus RNA1, a VEE GP helper (e.g. seePushko, et. al. 1997, ibid.) and a VEE RNA replicon expressing the HIVgag protein (see Olmsted et al., WO 02/03917). When the FHV RNA 1 and 2are used, recombinant alphavirus particle yields were 4.3×10⁶ per ml.When the NoV RNA 1 and 2 are used, particle yields were approximately2×10⁵ per ml.

Example 9

[0177] Production of Replicon Particles Using DNA or RNA Helpers

[0178] The replicon RNA and helper nucleic acids can be introduced tothe helper cell by one or more of several different techniques generallyknown to the art, e.g. electroporation, transfection (e.g. using calciumphosphate precipitation or nucleic acids deposited on microbeads ornanoparticles), lipid-mediated DNA uptake, micro- or nano-projectiles,stable transformation, by incorporation into a viral vector, such asadenovirus, SV40, poxvirus (e.g. modified vaccinia Ankara), nodavirus oradenoassociated virus, or by coating onto a virus vector, such asadenovirus. When using a combination of DNA helper nucleic acids and anRNA replicon, these molecules may be introduced at different times usingdifferent techniques. The difference in timing may provide a period oftime for recovery by the cells, and it may also allow optimization ofthe differing kinetics of expression from a DNA vector as compared withan RNA vector. The timing can be 30 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, or 24 hours between the introduction of each nucleic acidmolecule to the cells. The introduction of DNA helper(s) by a differentmeans and prior to the electroporation of replicon RNA (and helper RNA,as appropriate) does not have significant detrimental effects on theefficiency of the RNA electroporation.

[0179] Transfection of DNA Helpers

[0180] Lipid-Mediated

[0181] 293T, VERO or DF1 cells are first transfected with the DNA helperby lipofection using Fugene® 6 (Boehringer Mannheim, Indianapolis,Ind.). Cells are incubated in the presence of Fugene and DNA in mediumcontaining 10% FBS for 1 to 24 hours. After incubation, they are washedwith PBS and collected by trypsinization.

[0182] Electroporation

[0183] Transfection of DNA into Vero cells using commercially availablecationic lipids such as FuGene and Lipofectamine is typicallyinefficient (˜1% efficiency). While these methods may be sufficient forcertain applications, electroporation of DNA helpers into Vero cells isa preferred alternative approach. Various parameters of theelectroporation process can be optimized to enhance the efficiency ofDNA entry into Vero cells.

[0184] For example, it is preferable to use purified DNA constructs. Forexample, plasmid DNA containing a gene under the control of a CMVpromoter is first isolated using a high purity maxiprep system (e.g.Qiagen®; Promega Corporation, Madison, Wis.). Such initially purifed DNAis preferably further purified by phenol extraction in the presence ofethidium bromide and high salt (reference: Stemmer, W P, Biotechniques.June 1991; 10(6):726). This further purified DNA is resuspended innuclease free water prior to electroporation.

[0185] Another optimization step involves the culturing conditions forVero cells can be optimized for electroporation of DNA. Vero cells areinitially cultured in EMEM medium and grown to late log phase, thenharvested by trypsinization, washed twice with Invitrus® (Cell CultureTechnologies GmBh, Zurich, Switzerland), and resuspended in 800 μlInvitrus medium.

[0186] These Invitrus-bathed cells are combined with an optimized amountof purified DNA (typically, concentrations range from 10 μg to 200 μg)and then transferred to a 0.4 cm² gap cuvette. Electroporations isperformed using a Biorad Gene Pulser® (BioRad Laboratories, Inc.,Hercules, Calif.), with four pulses at 50 μF, over a range of voltages(500-700V). After electroporation, the Vero cells were seeded into 6well plates and incubated for 24 hours at 37° C. with 5% CO₂.

[0187] In determining the optimized parameters, expression of protein isfirst analyzed at 16 and 23 hours post-electroporation. The percentageof transfected cells, varying the above parameters, ranged from 1%-40%;an example of an optimized set of parameters is: 150 μg purified plasmidDNA and pulsing four times at 650V, 50 μF. In addition, there istypically an additional 5-10% increase in expression between 17 and 23hours post-electroporation.

[0188] Electroporation efficiency can also be enhanced by synchronizingthe Vero cells in a specific phase of the cell cycle. In one example,cells are synchronized in the G2/M phase with aphidicolin (1 mg/ml)prior to electroporation (see Golzio, M, Biochem Biophys Acta. June 200213:1563(1-2):23-8). These synchronized cells are handled as describedabove and combined with 50 μg of plasmid DNA prior to electroporation.In this example, aphidicolin-treated cells show a two-fold increase inthe percentage of cells transfected, as compared to untreated(unsynchronized) cells.

[0189] Electroporation of Alphavirus Replicon RNA

[0190] After introduction of the DNA helpers of this invention, 1.2×10⁷cells are then electroporated in the presence of a replicon RNA andappropriate helper RNAs (if required) under the following conditions:450V (293T) or 850V (VERO and DF1) and 25 uF in 0.4 cm gapelectroporation cuvettes (pulse 3×'s with 4 sec. between pulses).Electroporated cells are then allowed to recover for 10 min. beforeseeding into 25 mls medium containing 10% FBS.

[0191] When using RNA replicon and RNA helper combinations, all RNAs maybe co-electroporated into the helper cell at the same time. Methods forRNA electroporation are as described above. In an alternativeembodiment, when introducing the DNA helpers to the helper cell viaelectroporation, the replicon RNA may be co-electroporated with the DNAhelpers.

1 49 1 31 DNA Artificial Sequence Description of Artificial Sequence;NOTE = Synthetic Construct 1 ctagctagct atgtcactag tgaccaccat g 31 2 27DNA Artificial Sequence Description of Artificial Sequence; NOTE =Synthetic Construct 2 gggccctcaa ttatgtttct ggttggt 27 3 53 DNAArtificial Sequence Description of Artificial Sequence; NOTE = SyntheticConstruct 3 gcagagctgg tttagtgaac cgtataggcg gcgcatgaga gaagcccaga cca53 4 26 DNA Artificial Sequence Description of Artificial Sequence; NOTE= Synthetic Construct 4 gctagcgctc ttcccttttt tttttt 26 5 29 DNAArtificial Sequence Description of Artificial Sequence; NOTE = SyntheticConstruct 5 gctctagaat gttcccgttc cagccaatg 29 6 52 DNA ArtificialSequence Description of Artificial Sequence; NOTE = Synthetic Construct6 gcgtcgacgt cttggccata gcggccgcgg ttacagacac atggtggtca ct 52 7 91 DNAArtificial Sequence Description of Artificial Sequence; NOTE = SyntheticConstruct 7 cgggtcggca tggcatctcc acctcctcgc ggtccgacct gggcatccgaaggaggacgt 60 gtccactcg gatggctaag ggagagctcg c 1 8 99 DNA ArtificialSequence Description of Artificial Sequence; NOTE = Synthetic Construct8 tcgagcgagc tctcccttag ccatccgagt ggacgacgtc ctccttcgga tgcccaggtc 60ggaccgcgag gaggtggaga tgccatgccg acccgggcc 99 9 26 DNA ArtificialSequence Description of Artificial Sequence; NOTE = Synthetic Construct9 tagttattaa tagtaatcaa ttacgg 26 10 53 DNA Artificial SequenceDescription of Artificial Sequence; NOTE = Synthetic Construct 10tggtctgggc ttctctcatg cgccgcctat acggttcact aaaccagctc tgc 53 11 30 DNAArtificial Sequence Description of Artificial Sequence; NOTE = SyntheticConstruct 11 ggcgcgccgt cctccgatcg ttgtcagaag 30 12 36 DNA ArtificialSequence Description of Artificial Sequence; NOTE = Synthetic Construct12 ggcgcgcctc cgtcaaccgc gtatacatcc tggtaa 36 13 31 DNA ArtificialSequence Description of Artificial Sequence; NOTE = Synthetic Construct13 ggcgcgccat gtcactagtg accaccatgt g 31 14 23 DNA Artificial SequenceDescription of Artificial Sequence; NOTE = Synthetic Construct 14ctcgtaggcg ccggcgcctg cgg 23 15 29 DNA Artificial Sequence Descriptionof Artificial Sequence; NOTE = Synthetic Construct 15 ggcgcgccaattccgcccct ctccctccc 29 16 29 DNA Artificial Sequence Description ofArtificial Sequence; NOTE = Synthetic Construct 16 ggcgcgcctt atcatcgtgtttttcaaag 29 17 27 DNA Artificial Sequence Description of ArtificialSequence; NOTE = Synthetic Construct 17 gctagcaatt ccgcccctct ccctccc 2718 27 DNA Artificial Sequence Description of Artificial Sequence; NOTE =Synthetic Construct 18 gctagcttat catcgtgttt ttcaaag 27 19 29 DNAArtificial Sequence Description of Artificial Sequence; NOTE = SyntheticConstruct 19 cgacgcgtat gttcccgttc cagccaatg 29 20 30 DNA ArtificialSequence Description of Artificial Sequence; NOTE = Synthetic Construct20 gcacgcgttt acagacacat ggtggtcact 30 21 36 DNA Artificial SequenceDescription of Artificial Sequence; NOTE = Synthetic Construct 21cctgccatgg tataaatgtt cccgttccaa ccaatg 36 22 27 DNA Artificial SequenceDescription of Artificial Sequence; NOTE = Synthetic Construct 22cctgccatgg ccccgttcca accaatg 27 23 28 DNA Artificial SequenceDescription of Artificial Sequence; NOTE = Synthetic Construct 23gtctagaatg ttcccgttcc aaccaatg 28 24 29 DNA Artificial SequenceDescription of Artificial Sequence; NOTE = Synthetic Construct 24cgggatcccc attgctcgca gttctccgg 29 25 62 DNA Artificial SequenceDescription of Artificial Sequence; NOTE = Synthetic Construct 25cgggatccga aaacctgtat tttcagggca tgtcactagt gaccaccatg tgtctgctcg 60 cc62 26 26 DNA Artificial Sequence Description of Artificial Sequence;NOTE = Synthetic Construct 26 cgatatctca attatgtttc tggttg 26 27 28 DNAArtificial Sequence Description of Artificial Sequence; NOTE = SyntheticConstruct 27 gtctagaatg tccctagtga ccaccatg 28 28 28 DNA ArtificialSequence Description of Artificial Sequence; NOTE = Synthetic Construct28 gcgctagcgt caattatgtt tctggttg 28 29 28 DNA Artificial SequenceDescription of Artificial Sequence; NOTE = Synthetic Construct 29gtctagaatg ttcccgttcc aaccaatg 28 30 20 DNA Artificial SequenceDescription of Artificial Sequence; NOTE = Synthetic Construct 30caatcgccgc gagttctatg 20 31 34 DNA Artificial Sequence Description ofArtificial Sequence; NOTE = Synthetic Construct 31 tttcggaccg tctctacggtggtcctaaat agtc 34 32 20 DNA Artificial Sequence Description ofArtificial Sequence; NOTE = Synthetic Construct 32 ctggtcggat cattgggccc20 33 35 DNA Artificial Sequence Description of Artificial Sequence;NOTE = Synthetic Construct 33 cgggatccat gcggtttgat gcgggtgcat acatc 3534 31 DNA Artificial Sequence Description of Artificial Sequence; NOTE =Synthetic Construct 34 gctctagatt agccgtagag agttataggg g 31 35 30 DNAArtificial Sequence Description of Artificial Sequence; NOTE = SyntheticConstruct 35 tggcgcgccg ctcggaattc cccctctccc 30 36 29 DNA ArtificialSequence Description of Artificial Sequence; NOTE = Synthetic Construct36 aggcgcgcct tctatgtaag cagcttgcc 29 37 28 DNA Artificial SequenceDescription of Artificial Sequence; NOTE = Synthetic Construct 37ccgggaaaac agcattccag gtattaga 28 38 73 DNA Artificial SequenceDescription of Artificial Sequence; NOTE = Synthetic Construct 38tttttttttt tttttttttt tttttttttt tttttttttt tttttgaaat attaaaaaca 60aaatccgatt cgg 73 39 29 DNA Artificial Sequence Description ofArtificial Sequence; NOTE = Synthetic Construct 39 gacgggcccc ttgcccttcgtagcgacac 29 40 29 DNA Artificial Sequence Description of ArtificialSequence; NOTE = Synthetic Construct 40 gacgggccca gtttccaggt cagggtcgc29 41 33 DNA Artificial Sequence Description of Artificial Sequence;NOTE = Synthetic Construct 41 gacgggcccc cttcattttc ttgtccaatt cct 33 4234 DNA Artificial Sequence Description of Artificial Sequence; NOTE =Synthetic Construct 42 gacgggccct gcatacttat acaatctgtc cgga 34 43 35DNA Artificial Sequence Description of Artificial Sequence; NOTE =Synthetic Construct 43 gacgggcccc ggacagatac aatgatactt gtgct 35 44 30DNA Artificial Sequence Description of Artificial Sequence; NOTE =Synthetic Construct 44 gacgggcccg ccagatgcga aaacgctctg 30 45 30 DNAArtificial Sequence Description of Artificial Sequence; NOTE = SyntheticConstruct 45 gacgggcccg ccagatgcga aaacgctctg 30 46 29 DNA ArtificialSequence Description of Artificial Sequence; NOTE = Synthetic Construct46 gacgggccct acctcaaact gcgggaagc 29 47 32 DNA Artificial SequenceDescription of Artificial Sequence; NOTE = Synthetic Construct 47gacgggccct tttgggtagg taattggtct gg 32 48 23 DNA Artificial SequenceDescription of Artificial Sequence; NOTE = Synthetic Construct 48gacgggcccc tataactctc tac 23 49 20 DNA Artificial Sequence Descriptionof Artificial Sequence; NOTE = Synthetic Construct 49 gcaacgcggggaggcagaca 20

We claim:
 1. A recombinant DNA molecule for expressing alphavirusstructural proteins comprising a promoter directing the transcription ofRNA from a DNA sequence comprising, in order: (i) a first nucleic acidsequence encoding at least one alphavirus structural protein, (ii) asecond nucleic acid sequence encoding a ribozyme, (iii) a third nucleicacid sequence encoding an IRES, (iv) a fourth nucleic acid sequenceencoding at least one alphavirus structural protein, wherein at leastone alphavirus structural protein encoded by the fourth nucleic acidsequence is not encoded by the first nucleic acid sequence.
 2. Therecombinant DNA molecule of claim 1 wherein the first and fourth nucleicacid sequences together encode all alphavirus structural proteins. 3.The recombinant DNA molecule of claim 1 wherein the promoter is an RNApolymerase II promoter.
 4. The recombinant DNA molecule of claim 1wherein the first nucleic acid sequence encodes the alphavirusglycoprotein E1 and the fourth nucleic acid sequence encodes alphaviruscapsid protein.
 5. The recombinant DNA molecule of claim 1 wherein thefirst nucleic acid sequence encodes the alphavirus glycoprotein E2 andthe fourth nucleic acid sequence encodes alphavirus capsid protein. 6.The recombinant DNA molecule of claim 2 wherein the first nucleic acidsequence encodes alphavirus glycoproteins E1 and E2 and the fourthnucleic acid sequence encodes alphavirus capsid protein.
 7. Therecombinant DNA molecule of claim 2 wherein the first nucleic acidsequence encodes alphavirus capsid protein and the fourth nucleic acidsequence encodes alphavirus glycoproteins E1 and E2.
 8. The recombinantDNA molecule of claim 1 wherein the alphavirus is selected from thegroup consisting of VEE, Sindbis, TR339, S.A.AR86, Semliki Forest Virus,and Ross River Virus.
 9. The recombinant DNA molecule of claim 1 whereinthe IRES is derived from a gene selected from the group consisting ofvertebrate genes, invertebrate genes, genes of a virus that infectsvertebrate cells, and genes from a virus that infects insect cells. 10.A recombinant DNA molecule for expressing alphavirus structural proteinscomprising a promoter directing the transcription of RNA from a DNAsequence comprising, in order: (i) a first nucleic acid sequenceencoding a 5′ alphavirus replication recognition sequence, (ii) a secondnucleic acid sequence encoding an RNA sequence that promotestranscription of a protein coding RNA sequence; (iii) a third nucleicacid sequence encoding at least one alphavirus structural protein, (iv)a fourth nucleic acid sequence encoding a 3′ alphavirus replicationrecognition sequence (v) a fifth nucleic acid sequence encoding aribozyme, (vi) a sixth nucleic acid sequence encoding an IRES, (vii) aseventh nucleic acid sequence encoding at least one alphavirusstructural protein, wherein at least one alphavirus structural proteinencoded by the seventh nucleic acid sequence is not encoded by the thirdnucleic acid sequence.
 11. A recombinant DNA molecule for expressingalphavirus structural proteins comprising a promoter directing thetranscription of RNA from a DNA sequence comprising, in order: (i) afirst nucleic acid sequence encoding a 5′ alphavirus replicationrecognition sequence, (ii) a second nucleic acid sequence encoding anIRES, (iii) a third nucleic acid sequence encoding at least onealphavirus structural protein, (iv) a fourth nucleic acid sequenceencoding a 3′ alphavirus replication recognition sequence, (v) a fifthnucleic acid sequence encoding a ribozyme, (vi) a sixth nucleic acidsequence encoding an IRES, (vii) a seventh nucleic acid sequenceencoding at least one alphavirus structural protein, wherein at leastone alphavirus structural protein encoded by the seventh nucleic acidsequence is not encoded by the third nucleic acid sequence.
 12. Therecombinant DNA molecule of claim 10 or 11 wherein the third and theseventh nucleic acid sequences together encode all alphavirus structuralproteins.
 13. The recombinant DNA molecule of claim 10 or 11 wherein thethird nucleic acid sequence encodes alphavirus glycoproteins E1 and E2and the seventh nucleic acid sequence encodes alphavirus capsid protein.14. The recombinant DNA molecule of claim 10 or 11 wherein the thirdnucleic acid sequence encodes alphavirus capsid protein and the seventhnucleic acid sequence encodes alphavirus glycoproteins E1 and E2. 15.The recombinant DNA molecule of claim 10 wherein the second nucleic acidsequence is an alphavirus subgenomic promoter.
 16. The recombinant DNAmolecule of claim 10 or 11 wherein the first nucleic acid sequenceconsists of a minimal 5′ alphavirus replication recognition sequence.17. The recombinant DNA molecule of claim 10 or 11 wherein the promoteris an RNA polymerase II promoter.
 18. A method of producing infectious,replication-defective alphavirus replicon particles comprisingintroducing into a population of cells (i) a recombinant DNA molecule ofany one of claim 2, 6, 7, 10, or 11 encoding all the alphavirusstructural proteins, and (ii) an alphavirus replicon RNA encoding atleast one heterologous RNA, such that infectious, replication-defectivealphavirus replicon particles are produced.
 19. A method of producinginfectious, replication-defective alphavirus replicon particlescomprising introducing into a population of cells (i) two recombinantDNA molecules from any combination of claim 1, 4, 5, 10, or 11, and (ii)an alphavirus replicon RNA encoding at least one heterologous RNA,wherein the two recombinant molecules together encode all alphavirusstructural proteins, such that infectious, replication-defectivealphavirus replicon particles are produced.
 20. A recombinant DNAmolecule for expressing alphavirus structural proteins comprising apromoter directing the transcription of RNA from a DNA sequencecomprising, in order: (i) a first nucleic acid sequence encoding anIRES, (ii) a second nucleic acid sequence encoding at least onealphavirus structural protein, (iii) a third nucleic acid sequenceencoding a ribozyme, (iv) a fourth nucleic acid sequence encoding anIRES, (v) a fifth nucleic acid sequence encoding at least one alphavirusstructural protein, wherein at least one alphavirus structural proteinencoded by the fifth nucleic acid sequence is not encoded by the secondnucleic acid sequence.
 21. The recombinant DNA molecule of claim 20wherein the second and fifth nucleic acid sequences together encode allalphavirus structural proteins.
 22. The recombinant DNA molecule ofclaim 20 wherein the promoter is for an RNA polymerase selected from thegroup consisting of T7, T3, SP6 and eukaryotic and viral RNA polymeraseIIs.
 23. A method of producing infectious replication-defectivealphavirus replicon particles comprising introducing into a populationof cells (i) one or more recombinant DNA molecules of claim 20, and (ii)an alphavirus replicon RNA encoding at least one heterologous RNAwherein the recombinant DNA molecules encode all alphavirus structuralproteins such that infectious replication-defective alphavirus repliconparticles are produced.
 24. The method of claim 23 further comprisingintroducing into the population of cells a DNA dependent RNA polymerasethat recognizes the promoter.
 25. The method of claim 23 wherein the RNApolymerase is introduced to the population of cells on a separateplasmid encoding an RNA polymerase II promoter operably linked to anddirecting the expression of a nucleic acid sequence encoding the RNApolymerase.
 26. The method of claim 23 wherein the population of cellsis derived from a cell line stably transformed with a gene encoding theRNA polymerase and wherein the RNA polymerase is introduced byexpression from the stably transformed gene.
 27. The method of any oneof claim 24, 25 or 26 wherein the RNA polymerase is T7 and the promoteris the T7 promoter.
 28. A recombinant DNA molecule for expressingalphavirus structural proteins comprising: (i) a DNA dependent RNApolymerase promoter, (ii) an IRES, (iii) a nucleic acid sequenceencoding an alphavirus capsid protein, which is modified to remove theactive site of the autoprotease, (iv) a non-autocatalytic proteaserecognition site, and (v) a nucleic acid sequence encoding at least onealphavirus glycoprotein.
 29. The recombinant DNA molecule of claim 28wherein the RNA polymerase promoter is the T7 promoter.
 30. Therecombinant RNA molecule of claim 28 wherein at least one of the nucleicacid sequences of (ii) and (iv) include an attenuating mutation.
 31. Therecombinant RNA molecule of claim 28 wherein the non-autocatalyticprotease recognition site is from tobacco etch virus.
 32. A method ofproducing infectious, replication-defective alphavirus repliconparticles comprising introducing into a population of cells (i) one ormore recombinant DNA molecules of claim 28, (ii) an RNA polymerase thatrecognizes the DNA dependent RNA polymerase promoter, (iii) a proteasethat recognizes the non-autocatalytic protease recognition site, and(iv) an alphavirus replicon RNA encoding at least one heterologous RNAsuch that infectious, replication-defective alphavirus repliconparticles are produced.
 33. A recombinant nucleic acid moleculecomprising, in order: (i) a first nucleic acid sequence encoding a 5′alphavirus replication recognition sequence, (ii) a second nucleic acidencoding alphavirus nonstructural proteins nsp1, nsp2, and nsp3; (iii) atranscriptional promoter, (iv) a nucleic acid encoding at least oneheterologous gene of interest, (v) an IRES, (vi) a third nucleic acidencoding an alphavirus nonstructural protein nsp4, and (vii) a fourthnucleic acid encoding a 3′ alphavirus replication recognition sequence.34. A recombinant nucleic acid comprising, in order: (i) a first nucleicacid sequence encoding a 5′ alphavirus replication recognition sequence,(ii) a second nucleic acid encoding alphavirus nonstructural proteinsnsp 1, nsp 2, and nsp3; (iii) an IRES, (iv) a nucleic acid encoding atleast one heterologous gene of interest, (v) an IRES, (vi) a thirdnucleic acid encoding the alphavirus nonstructural protein nsp4, and(vii) a fourth nucleic acid encoding a 3′ alphavirus replicationrecognition sequence.
 35. The recombinant nucleic acid of claim 33wherein the nucleic acid is RNA.
 36. The recombinant nucleic acid ofclaim 34 wherein the nucleic acid is RNA.
 37. The recombinant nucleicacid of claim 33 or 34 wherein the first, second, third and fourthnucleic acids are derived from VEE, Semliki Forest Virus, or SindbisVirus.
 38. An alphavirus vector construct comprising a 5′ promoteroperably linked to a cDNA of the RNA of claim
 35. 39. An alphavirusvector construct comprising a 5′ promoter operably linked to a cDNA ofthe RNA of claim
 36. 40. A composition comprising a population ofinfectious, defective, alphavirus replicon particles, wherein eachparticle contains an alphavirus replicon RNA comprising the nucleic acidof claim 35, and the population has no detectable replication-competentvirus, as measured by passage on cell cultures.
 41. A compositioncomprising a population of infectious, defective, alphavirus repliconparticles, wherein each particle contains an alphavirus replicon RNAcomprising the nucleic acid of claim 36, and the population has nodetectable replication-competent virus, as measured by passage on cellcultures.
 42. The recombinant nucleic acid of claim 33 wherein thetranscriptional promoter is an alphavirus 26S subgenomic promoter.
 43. Arecombinant DNA molecule for expressing alphavirus structural proteinscomprising a promoter for directing the transcription of RNA from a DNAsequence operably linked to a DNA sequence encoding a completealphavirus structural polyprotein-coding sequence, with the proviso thatthe DNA sequence does not encode alphaviral 5′ or 3′ replicationrecognition sequences or an alphavirus subgenomic promoter.
 44. A helpercell for expressing an infectious, replication-defective, alphavirusreplicon particle comprising, in an alphavirus-permissive cell, (i) oneor more recombinant nucleic acid molecules selected from any of claim 1,10, 11, 20, or 28; and (ii) an alphavirus replicon RNA encoding at leastone heterologous RNA, wherein the one or more recombinant nucleic acidstogether encode all alphavirus structural proteins which assembletogether into the alphavirus replicon particles.
 45. A helper cell forexpressing an infectious, replication-defective, alphavirus repliconparticle, selected from the group consisting of 293, 293T, BHK, Vero,CHO, CEF and DF-1, comprising: (i) one or more recombinant nucleic acidmolecules selected from any of claim 1,10, 11, 20, or 28; and (ii) analphavirus replicon RNA encoding at least one heterologous RNA, whereinthe one or more recombinant nucleic acids together encode all alphavirusstructural proteins which assemble together into the alphavirus repliconparticles.
 46. A recombinant RNA molecule comprising, in order, a 5′alphavirus replication recognition sequence, a promoter, a nucleic acidencoding at least one alphavirus structural protein, and a 3′ alphaviralreplication recognition sequence, wherein the promoter is operablylinked to the nucleic acid sequence encoding at least one alphavirusstructural protein, wherein the transcription-initiating sequence andthe RNA polymerase recognition sequence are recognized by thenonstructural viral proteins of Venezuelan Equin Encephalitis virus, andwherein the transcription-initiating sequence and the RNA polymeraserecognition sequence are derived from a virus other than the alphavirusencoding the structural protein.
 47. The recombinant RNA molecule ofclaim 46 wherein the alphavirus structural protein is selected from thegroup consisting of capsid, capsid-E1, capsid-E2, or E1-E2.
 48. Therecombinant RNA molecule of claim 46 wherein the nucleic acid sequenceencoding the at least one alphavirus structural protein comprises one ormore attenuating mutations.
 49. The recombinant RNA molecule of claim 46wherein the nucleic acid sequence encoding at least one alphavirusstructural protein encodes all the structural proteins of saidalphavirus.
 50. The recombinant RNA molecule of claim 46 wherein the 5′and 3′ replication recognition sequences are derived from a virusselected from the group consisting of Sindbis Virus, Semliki ForestVirus, and Ross River virus
 51. A cDNA molecule encoding the recombinantRNA molecule of any one of claim 46, 47, 48, 49, or
 50. 52. A method ofmaking infectious, defective alphavirus particles, comprisingintroducing into a population of cells the following: (a) a recombinantRNA molecule according to claim 47; (b) an alphavirus replicon RNA; (c)a second recombinant RNA molecule according to claim 47; wherein therecombinant RNA molecules of (a) and (c) express all of the alphavirusstructural proteins, producing said alphavirus particles in the cells,and collecting said alphavirus particles from the cells.
 53. The methodaccording to claim 52, wherein the 5′ and 3′ sequences of the alphavirusreplicon RNA of (b) are from a different alphavirus than the 5′ and 3′sequences in the molecule of (a).
 54. An alphavirus structural proteinexpression system, comprising two RNA molecules, wherein (a) a first RNAencodes sequence for viral replicase proteins, and (b) a secondrecombinant RNA encodes sequences for (i) the 5′ replication recognitionsequence for a replication complex comprising the viral replicaseproteins of (a), (ii) one or more alphavirus structural proteins, and(iii) the 3′ replication recognition sequence for the replicationcomplex comprising the viral replicase proteins of (a), wherein, whenthe first RNA and the second RNA are introduced into a helper cell, thefirst RNA replicates the second RNA, then the second RNA is translatedto produce one or more alphavirus structural proteins.
 55. Theexpression system of claim 54 wherein the viral replicase proteins arefrom a nodavirus.
 56. The expression system of claim 55 wherein thenodavirus is selected from the group consisting of flock house virus andnodamura virus.
 57. The expression system of claim 54 wherein thealphavirus structural protein is capsid.
 58. The expression system ofclaim 54 wherein the alphavirus is selected from the group consisting ofVEE, SA.A.R.86, TR339, Sindbis, Ross River, and Semliki Forest.
 59. Theexpression system of claim 54 wherein the RNA molecules are introducedto the helper cell by a method selected from the group consisting ofvirus and electroporation.
 60. A helper cell containing the expressionsystem of claim
 54. 61. The expression system of claim 59 wherein thevirus is selected from the group consisting of adenovirus, vaccinia,poxvirus, SV-40, adeno-associated virus, retrovirus, nodavirus,picornavirus, vesicular stomatitis virus, and baculoviruses withmammalian pol II promoters.
 62. The expression system of claim 54wherein the host cell is selected from the group of cells consisting ofVero, Baby Hamster Kidney, Chinese Hamster Ovary, chicken embryofibroblasts, DF-1, 293, 293T, and mosquito cells.
 63. A method formaking infectious, replication-defective alphavirus replicon particles,comprising introducing into a population of cells the following: (a) analphavirus structural protein expression system according to claim 54;(b) an alphavirus replicon RNA encoding at least one heterologous RNA;(c) a recombinant RNA molecule capable of expressing in the cells thealphavirus structural glycoprotein; producing said alphavirus repliconparticles in the cells, and collecting said alphavirus repliconparticles from the cells.
 64. A method for making infectious,replication-defective alphavirus replicon particles, comprisingintroducing into a population of cells the following: (a) an alphavirusstructural protein expression system according to claim 54, and (b) analphavirus replicon RNA encoding at least one heterologous RNA; whereinthe alphavirus structural protein expression system expresses all thealphavirus structural proteins, producing said alphavirus repliconparticles in the cells, and collecting said alphavirus repliconparticles from the cells.
 65. The method according to claim 63 or 64,wherein the alphavirus structural proteins are selected from the groupconsisting of VEE, Sindbis, S.A.AR 86, Semliki Forest Virus and RossRiver Virus.